Innate Responses to the Former COVID-19 Vaccine Candidate CVnCoV and Their Relation to Reactogenicity and Adaptive Immunogenicity.

Vaccines are highly effective at preventing severe coronavirus disease (COVID-19). With mRNA vaccines, further research is needed to understand the association between immunogenicity and reactogenicity, which is defined as the physical manifestation of an inflammatory response to a vaccination. This study analyzed the immune response and reactogenicity in humans, post immunization, to the former SARS-CoV-2 mRNA investigational vaccine CVnCoV (CV-NCOV-001 and CV-NCOV-002 clinical trials). Immunogenicity was investigated using whole-blood RNA sequencing, serum cytokine levels, and SARS-CoV-2-specific antibodies. The T cell responses in peripheral blood were assessed using intracellular cytokine staining (ICS) and high-dimensional profiling in conjunction with SARS-CoV-2 antigen-specificity testing via mass cytometry. Reactogenicity was graded after participants' first and second doses of CVnCoV using vaccine-related solicited adverse events (AEs). Finally, a Spearman correlation was performed between reactogenicity, humoral immunity, and serum cytokine levels to assess the relationship between reactogenicity and immunogenicity post CVnCoV vaccination. Our findings showed that the gene sets related to innate and inflammatory immune responses were upregulated one day post CVnCoV vaccination, while the gene sets related to adaptive immunity were upregulated predominantly one week after the second dose. The serum levels of IFNα, IFNγ, IP-10, CXCL11, IL-10, and MCP-1 increased transiently, peaking one day post vaccination. CD4+ T cells were induced in all vaccinated participants and low frequencies of CD8+ T cells were detected by ex vivo ICS. Using mass cytometry, SARS-CoV-2 spike-specific CD8+ T cells were induced and were characterized as having an activated effector memory phenotype. Overall, the results demonstrated a positive correlation between vaccine-induced systemic cytokines, reactogenicity, and adaptive immunity, highlighting the importance of the balance between the induction of innate immunity to achieve vaccine efficacy and ensuring low reactogenicity.

CCR9 signaling in dendritic cells drives the differentiation of Foxp3+ Tregs and suppresses the allergic IgE response in the gut.

The chemokine receptor CCR9 and its only known ligand CCL25 play an important role in gut inflammation and autoimmune colitis. The function of CCR9-CCL25 in the migration of immune cells is well characterized. However, its role in the immune cell differentiation is mostly not known. Using dextran sodium sulfate (DSS)-induced gut inflammation model, we showed that CCR9+ dendritic cells (DCs) specifically CD11b- CD103+ DCs were significantly increased in the gut-associated lymphoid tissues (GALT) compared to control mice. These CCR9+ DCs express lower MHC II and CD86 molecules and had regulatory surface markers (FasL and latency-associated peptide, LAP) in the GALT. In the presence of CCL25, CCR9+ DCs promoted in vitro differentiation of Foxp3+ regulatory CD4+ T cells (Tregs). CCL25-induced differentiation of Tregs was due to intrinsic signaling in the DCs but not through CD4+ T cells, which was driven by the production of thymic stromal lymphopoietin (TSLP) and not IL-10. Furthermore, adoptive transfer of CCR9+ DCs in C57BL/6 mice promoted Tregs but reduced the Th17 cells in the GALT, and also suppressed the OVA-specific gut-allergic response. Our results suggest CCR9+ DCs have a regulatory function and may provide a new cellular therapeutic strategy to control gut inflammation and allergic immune reaction.

CCR6 signaling inhibits suppressor function of induced-Treg during gut inflammation.

CCR6 is a G protein-coupled receptor (GPCR) that binds to a specific chemokine, CCL20. The role of CCR6-CCL20 is very well studied in the migration of immune cells, but the non-chemotaxis functions of CCR6 signaling were not known. Here, we show that during gut inflammation, the frequency of Foxp3+CD4+ T cells (Tregs) reduced in the secondary lymphoid tissues and CCR6+ Tregs enhanced the expression of RORγt. The peripheral blood mononuclear cells (PBMCs) of ulcerative colitis (UC) patients showed lower percentages of Foxp3+CD4+ T cells, as compared to healthy individuals, with CCR6+ Tregs showing higher RORγt expression as compared to CCR6-Tregs. CCL20 inhibited the TGF-ß1-induced Treg (iTreg) differentiation and directed them towards the pathogenic Th17-lineage in a CCR6-dependent manner. The iTreg that differentiated in the presence of CCL20 showed lower surface expression of suppressor molecules such as CD39, CD73 and FasL, and had impaired suppressive function. Furthermore, CCR6 signaling induced phosphorylation of Akt, mTOR, and STAT3 molecules in T cells. In conclusion, we have identified a new role of CCR6 signaling in the differentiation of iTregs during inflammation and gut autoimmunity.

Synergy between the classical and alternative pathways of complement is essential for conferring effective protection against the pandemic influenza A(H1N1) 2009 virus infection.

The pandemic influenza A(H1N1) 2009 virus caused significant morbidity and mortality worldwide thus necessitating the need to understand the host factors that influence its control. Previously, the complement system has been shown to provide protection during the seasonal influenza virus infection, however, the role of individual complement pathways is not yet clear. Here, we have dissected the role of intact complement as well as of its individual activation pathways during the pandemic influenza virus infection using mouse strains deficient in various complement components. We show that the virus infection in C3-/- mice results in increased viral load and 100% mortality, which can be reversed by adoptive transfer of naïve wild-type (WT) splenocytes, purified splenic B cells, or passive transfer of immune sera from WT, but not C3-/- mice. Blocking of C3a and/or C5a receptor signaling in WT mice using receptor antagonists and use of C3aR-/- and C5aR-/- mice showed significant mortality after blocking/ablation of C3aR, with little or no effect after blocking/ablation of C5aR. Intriguingly, deficiency of C4 and FB in mice resulted in only partial mortality (24%-32%) suggesting a necessary cross-talk between the classical/lectin and alternative pathways for providing effective protection. In vitro virus neutralization experiments performed to probe the cross-talk between the various pathways indicated that activation of the classical and alternative pathways in concert, owing to coating of viral surface by antibodies, is needed for its efficient neutralization. Examination of the virus-specific complement-binding antibodies in virus positive subjects showed that their levels vary among individuals. Together these results indicate that cooperation between the classical and alternative pathways not only result in efficient direct neutralization of the pandemic influenza virus, but also lead to the optimum generation of C3a, which when sensed by the immune cells along with the antigen culminates in generation of effective protective immune responses.

Role of chemokine receptors and intestinal epithelial cells in the mucosal inflammation and tolerance.

The intestinal epithelial lining is a very dynamic interface, where multiple interactions occur with the external world. The intestinal epithelial barrier is continuously exposed to a huge load of commensal microorganisms, food-borne antigens, as well as invading enteropathogens. Intestinal epithelial cells (IECs) and underlying immune cells are the main players in maintaining the delicate balance between gut tolerance and inflammation. IECs deferentially express the variety of chemokines and chemokine receptors, and these receptor-ligand interactions not only mediate the infiltration and activation of immune cells but also switch on the survival cascades in IECs. In this review, we discussed how chemokine-chemokine receptor-induced interactions play a central role to coordinate the interplay between IECs and gut immune cells to maintain homeostasis or elicit gut inflammation. Furthermore, we discussed how chemokines and chemokine receptors were used as a target for developing new drugs and therapies to control gut inflammation and autoimmunity.

IL-10 from marginal zone precursor B cells controls the differentiation of Th17, Tfh and Tfr cells in transplantation tolerance.

B cells are known to control CD4T cell differentiation in secondary lymphoid tissues. We hypothesized that IL-10 expression by marginal zone precursor (MZP) regulatory B cells controls the differentiation and positioning of effector and regulatory T cells during tolerization. Costimulatory blockade with donor-specific transfusion (DST) and anti-CD40L mAb in C57BL/6 mice induced tolerance to allogeneic cardiac allograft. B cell depletion or IL-10 deficiency in B cells prevented tolerance, resulting in decreased follicular regulatory CD4(+) T cells (Tfr) and increased IL-21 expression by T follicular helper (Tfh) cells in the B cell and T cell zones. IL-21 acted with IL-6 to induce CCR6(+) Th17 that caused rejection. Deficiency or blockade of IL-6, IL-21, IL-21R, or CCR6 prevented B cell depletion-induced acute cellular rejection; while agonistic mCCL20-Ig induced rejection. Adoptive transfer of IL-10(+/+) MZP in tolerogen treated CD19-Cre(+/-):IL-10(fl/fl) mice rescued the localization of Tfh and Tfr cells in the B cell follicle and prevented allograft rejection. MZP B cell IL-10 is necessary for tolerance and controls the differentiation and position of Th17, Tfh and Tfr cells in secondary lymphoid tissues. This has implications for understanding tolerance induction and how B cell depletion may prevent tolerance.

Intratumoral natural killer cells show reduced effector and cytolytic properties and control the differentiation of effector Th1 cells.

Natural killer (NK) cells are known to have effector and cytolytic properties to kill virus infected or tumor cells spontaneously. Due to these properties, NK cells have been used as an adoptive cellular therapy to control tumor growth in various clinical trials but have shown limited clinical benefits. This indicates that our knowledge about phenotypic and functional differences in NK cells within the tumor microenvironment and secondary lymphoid tissues is incomplete. In this work, we report that B16F10 cell-induced melanoma recruits the CD11b+CD27+ subset of NK cells at a very early stage during tumor progression. These intratumoral NK cells showed increased expression of CD69, reduced inhibitory receptor KLRG1, and decreased proliferative ability. As compared to splenic NK cells, intratumoral NK cells showed decreased expression of activating receptors NKG2D, Ly49D and Ly49H; increased inhibitory receptors, NKG2A and Ly49A; decreased cytokines IFNγ and GM-CSF; decreased cytokine receptors IL-21R, IL-6Rα, and CD122 expression. Depletion of NK cells led to decrease peripheral as well as intratumoral effector CD4+T-bet+ cells (Th1), and increased tumor growth. Furthermore, purified NK cells showed increased differentiation of Th1 cells in an IFNγ-dependent manner. Anti-NKG2D in the culture promoted differentiation of effector Th1 cells. Collectively, these observations suggest that intratumoral NK cells possess several inhibitory functions that can be partly reversed by signaling through the NKG2D receptor or by cytokine stimulation, which then leads to increased differentiation of effector Th1 cells.

Interleukin-10 From Marginal Zone Precursor B-Cell Subset Is Required for Costimulatory Blockade-Induced Transplantation Tolerance.

BACKGROUND: Blocking CD40-CD40L costimulatory signals induces transplantation tolerance. Although B-cell depletion prevents alloantibody formation, nonhumoral functions of B cells in tolerance have not been well characterized. We investigated whether specific subsets of B cell or B cell-derived interleukin (IL)-10 contribute to tolerance. METHODS: Wild type C57BL/6, or B cell-specific interleukin (IL)-10 (CD19-Cre::IL-10) mice, received vascularized BALB/c cardiac allografts. BALB/c donor-specific splenocyte transfusion and anti-CD40L monoclonal antibody were used as tolerogen. B cells were depleted with antimouse CD20 monoclonal antibody. Various B-cell subsets were purified and characterized by flow cytometry, reverse transcription polymerase chain reaction, and adoptive transfer. RESULTS: B-cell depletion prevented costimulatory blockade-induced allogeneic tolerance. Costimulatory blockade increased IL-10 in marginal zone precursor (MZP) B cells, but not other subsets. In particular, costimulatory blockade did not change other previously defined regulatory B-cell subsets (Breg), including CD5CD1d Breg or expression of TIM1 or TIM4 on these Breg or other Breg cell subsets. Costimulatory blockade also induced IL-21R expression in MZP B cells, and IL-21R MZP B cells expressed even more IL-10. B-cell depletion or IL-10 deficiency in B cells prevented tolerance in a cardiac allograft model, resulting in rapid acute cardiac allograft rejection. Adoptive transfer of wild type MZP B cells but not other subsets to B cell-specific IL-10 deficient mice prevented graft rejection. CONCLUSIONS: CD40 costimulatory blockade induces MZP B cell IL-10 which is necessary for tolerance. These observations have implications for understanding tolerance induction and how B cell depletion may prevent tolerance.

Role of miRNAs in CD4 T cell plasticity during inflammation and tolerance.

Gene expression is tightly regulated in a tuneable, cell-specific and time-dependent manner. Recent advancement in epigenetics and non-coding RNA (ncRNA) revolutionized the concept of gene regulation. In order to regulate the transcription, ncRNA can promptly response to the extracellular signals as compared to transcription factors present in the cells. microRNAs (miRNAs) are ncRNA (~22 bp) encoded in the genome, and present as intergenic or oriented antisense to neighboring genes. The strategic location of miRNA in coding genes helps in the coupled regulation of its expression with host genes. miRNA together with complex machinery called RNA-induced silencing complex (RISC) interacts with target mRNA and degrade the mRNA or inhibits the translation. CD4 T cells play an important role in the generation and maintenance of inflammation and tolerance. Cytokines and chemokines present in the inflamed microenvironment controls the differentiation and function of various subsets of CD4 T cells [Th1, Th2, Th17, and regulatory CD4 T cells (Tregs)]. Recent studies suggest that miRNAs play an important role in the development and function of all subsets of CD4 T cells. In current review, we focused on how various miRNAs are regulated by cell's extrinsic and intrinsic signaling, and how miRNAs affect the transdifferentiation of subsets of CD4 T cell and controls their plasticity during inflammation and tolerance.