Distinct T cell responsiveness to different COVID-19 vaccines and cross-reactivity to SARS-CoV-2 variants with age and CMV status.

Introduction: Accumulating evidence indicates the importance of T cell immunity in vaccination-induced protection against severe COVID-19 disease, especially against SARS-CoV-2 Variants-of-Concern (VOCs) that more readily escape from recognition by neutralizing antibodies. However, there is limited knowledge on the T cell responses across different age groups and the impact of CMV status after primary and booster vaccination with different vaccine combinations. Moreover, it remains unclear whether age has an effect on the ability of T cells to cross-react against VOCs. Methods: Therefore, we interrogated the Spike-specific T cell responses in healthy adults of the Dutch population across different ages, whom received different vaccine types for the primary series and/or booster vaccination, using IFNÉ£ ELISpot. Cells were stimulated with overlapping peptide pools of the ancestral Spike protein and different VOCs. Results: Robust Spike-specific T cell responses were detected in the vast majority of participants upon the primary vaccination series, regardless of the vaccine type (i.e. BNT162b2, mRNA-1273, ChAdOx1 nCoV-19, or Ad26.COV2.S). Clearly, in the 70+ age group, responses were overall lower and showed more variation compared to younger age groups. Only in CMV-seropositive older adults (>70y) there was a significant inverse relation of age with T cell responses. Although T cell responses increased in all age groups after booster vaccination, Spike-specific T cell frequencies remained lower in the 70+ age group. Regardless of age or CMV status, primary mRNA-1273 vaccination followed by BNT162b2 booster vaccination showed limited booster effect compared to the BNT162b2/BNT162b2 or BNT162b2/mRNA-1273 primary-booster regimen. A modest reduction in cross-reactivity to the Alpha, Delta and Omicron BA.1, but not the Beta or Gamma variant, was observed after primary vaccination. Discussion: Together, this study shows that age, CMV status, but also the primary-booster vaccination regimen influence the height of the vaccination-induced Spike-specific T cell response, but did not impact the VOC cross-reactivity.

Serological profiling of pneumococcal proteins reveals unique patterns of acquisition, maintenance and waning of antibodies throughout life.

Streptococcus pneumoniae is a leading cause of morbidity and mortality in children and older adults. Yet knowledge on the development of pneumococcal protein-specific antibody responses throughout life is limited. To investigate this, we measured serum IgG levels to 55 pneumococcal proteins in 11-month old infants (n=73), 24-month old children (n=101), parents (n=99), adults without children <6 years of age (n= 99) and older adults aged >60 years (n=100). Our findings revealed low IgG levels in infancy, with distinct development patterns peaking in adults. A decrease in levels was observed for 27 antigens towards older age. Adults and older adults had increased IgG levels during pneumococcal carriage and at increased exposure risk to S. pneumoniae. Carriage was a stronger predictor than exposure or age for antibody responses. These findings highlight the dynamic nature of naturally acquired humoral immunity to pneumococcal proteins throughout life, offering insights for age-targeted interventions.

Human CD34+-derived complete plasmacytoid and conventional dendritic cell vaccine effectively induces antigen-specific CD8+ T cell and NK cell responses in vitro and in vivo.

Allogeneic stem cell transplantation (alloSCT) can be curative for hemato-oncology patients due to effective graft-versus-tumor immunity. However, relapse remains the major cause of treatment failure, emphasizing the need for adjuvant immunotherapies. In this regard, post-transplantation dendritic cell (DC) vaccination is a highly interesting strategy to boost graft-versus-tumor responses. Previously, we developed a clinically applicable protocol for simultaneous large-scale generation of end-stage blood DC subsets from donor-derived CD34+ stem cells, including conventional type 1 and 2 DCs (cDC1s and cDC2s), and plasmacytoid DCs (pDCs). In addition, the total cultured end-product (DC-complete vaccine), also contains non-end-stage-DCs (i.e. non-DCs). In this study, we aimed to dissect the phenotypic identity of these non-DCs and their potential immune modulatory functions on the potency of cDCs and pDCs in stimulating tumor-reactive CD8+ T and NK cell responses, in order to obtain rationale for clinical translation of our DC-complete vaccine. The non-DC compartment was heterogeneous and comprised of myeloid progenitors and (immature) granulocyte- and monocyte-like cells. Importantly, non-DCs potentiated toll-like receptor-induced DC maturation, as reflected by increased expression of co-stimulatory molecules and enhanced cDC-derived IL-12 and pDC-derived IFN-α production. Additionally, antigen-specific CD8+ T cells effectively expanded upon DC-complete vaccination in vitro and in vivo. This effect was strongly augmented by non-DCs in an antigen-independent manner. Moreover, non-DCs did not impair in vitro DC-mediated NK cell activation, degranulation nor cytotoxicity. Notably, in vivo i.p. DC-complete vaccination activated i.v. injected NK cells. Together, these data demonstrate that the non-DC compartment potentiates DC-mediated activation and expansion of antigen-specific CD8+ T cells and do not impair NK cell responses in vitro and in vivo. This underscores the rationale for further clinical translation of our CD34+-derived DC-complete vaccine in hemato-oncology patients post alloSCT.

Lipid-loaded tumor-associated macrophages sustain tumor growth and invasiveness in prostate cancer.

Tumor-associated macrophages (TAMs) are correlated with the progression of prostatic adenocarcinoma (PCa). The mechanistic basis of this correlation and therapeutic strategies to target TAMs in PCa remain poorly defined. Here, single-cell RNA sequencing was used to profile the transcriptional landscape of TAMs in human PCa, leading to identification of a subset of macrophages characterized by dysregulation in transcriptional pathways associated with lipid metabolism. This subset of TAMs correlates positively with PCa progression and shorter disease-free survival and is characterized by an accumulation of lipids that is dependent on Marco. Mechanistically, cancer cell-derived IL-1ß enhances Marco expression on macrophages, and reciprocally, cancer cell migration is promoted by CCL6 released by lipid-loaded TAMs. Moreover, administration of a high-fat diet to tumor-bearing mice raises the abundance of lipid-loaded TAMs. Finally, targeting lipid accumulation by Marco blockade hinders tumor growth and invasiveness and improves the efficacy of chemotherapy in models of PCa, pointing to combinatorial strategies that may influence patient outcomes.

CD34+ progenitor-derived NK cell and gemcitabine combination therapy increases killing of ovarian cancer cells in NOD/SCID/IL2Rgnull mice.

Combining natural killer (NK) cell adoptive transfer with tumor-sensitizing chemotherapy is an attractive approach against recurrent ovarian cancer (OC), as OC is sensitive to NK cell-mediated immunity. Previously, we showed that CD34+ hematopoietic progenitor cell (HPC)-derived NK cells can kill OC cells in vitro and inhibit OC tumor growth in mice. Here, we investigated the potential of HPC-NK cell therapy combined with chemotherapeutic gemcitabine (used in recurrent OC patients) against OC. We examined the phenotypical, functional, and cytotoxic effects of gemcitabine on HPC-NK cells and/or OC cells in vitro and in OC-bearing mice. To this end, we treated OC cells and/or HPC-NK cells with or without gemcitabine and analyzed the phenotype, cytokine production, and anti-tumor reactivity. We found that gemcitabine did not affect the phenotype and functionality of HPC-NK cells, while on OC cells expression of NK cell activating ligands and death receptors was upregulated. Although gemcitabine pre-treatment of OC cells did not improve the functionality of HPC-NK cells, importantly, HPC-NK cells and gemcitabine additively killed OC cells in vitro. Similarly, combined HPC-NK cell and gemcitabine treatment additively decreased tumor growth in OC-bearing mice. Collectively, our results indicate that combination therapy of HPC-NK cells and gemcitabine results in augmented OC killing in vitro and in vivo. This provides a rationale for exploring this therapeutic strategy in patients with recurrent OC.

Single-cell profiling defines the prognostic benefit of CD39high tissue resident memory CD8+ T cells in luminal-like breast cancer.

Luminal-like breast cancer (BC) constitutes the majority of BC subtypes, but, differently from highly aggressive triple negative BC, is poorly infiltrated by the immune system. The quality of the immune infiltrate in luminal-like BCs has been poorly studied, thereby limiting further investigation of immunotherapeutic strategies. By using high-dimensional single-cell technologies, we identify heterogeneous behavior within the tissue-resident memory CD8+ T (Trm) cells infiltrating luminal-like tumors. A subset of CD127- CD39hi Trm cells, preferentially present in the tumor compared to the adjacent normal breast tissue or peripheral blood, retains enhanced degranulation capacity compared to the CD127+ CD39lo Trm counterpart ex vivo, and is specifically associated with positive prognosis. Nevertheless, such prognostic benefit is lost in the presence of highly-suppressive CCR8hi ICOShi IRF4+ effector Tregs. Thus, combinatorial strategies aiming at boosting Trm function and infiltration while relieving from Treg-mediated immunosuppression should be investigated to achieve proper tumor control in luminal-like BCs.

Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

Circulating mucosal-associated invariant T cells identify patients responding to anti-PD-1 therapy.

Immune checkpoint inhibitors are used for treating patients with metastatic melanoma. Since the response to treatment is variable, biomarkers are urgently needed to identify patients who may benefit from such therapy. Here, we combine single-cell RNA-sequencing and multiparameter flow cytometry to assess changes in circulating CD8+ T cells in 28 patients with metastatic melanoma starting anti-PD-1 therapy, followed for 6 months: 17 responded to therapy, whilst 11 did not. Proportions of activated and proliferating CD8+ T cells and of mucosal-associated invariant T (MAIT) cells are significantly higher in responders, prior to and throughout therapy duration. MAIT cells from responders express higher level of CXCR4 and produce more granzyme B. In silico analysis support MAIT presence in the tumor microenvironment. Finally, patients with >1.7% of MAIT among peripheral CD8+ population show a better response to treatment. Our results thus suggest that MAIT cells may be considered a biomarker for patients responding to anti-PD-1 therapy.

IRF4 instructs effector Treg differentiation and immune suppression in human cancer.

The molecular mechanisms responsible for the high immunosuppressive capacity of CD4+ Tregs in tumors are not well known. High-dimensional single-cell profiling of T cells from chemotherapy-naive individuals with non-small-cell lung cancer identified the transcription factor IRF4 as specifically expressed by a subset of intratumoral CD4+ effector Tregs with superior suppressive activity. In contrast to the IRF4- counterparts, IRF4+ Tregs expressed a vast array of suppressive molecules, and their presence correlated with multiple exhausted subpopulations of T cells. Integration of transcriptomic and epigenomic data revealed that IRF4, either alone or in combination with its partner BATF, directly controlled a molecular program responsible for immunosuppression in tumors. Accordingly, deletion of Irf4 exclusively in Tregs resulted in delayed tumor growth in mice while the abundance of IRF4+ Tregs correlated with poor prognosis in patients with multiple human cancers. Thus, a common mechanism underlies immunosuppression in the tumor microenvironment irrespective of the tumor type.

Single-Cell Sequencing of Mouse Heart Immune Infiltrate in Pressure Overload-Driven Heart Failure Reveals Extent of Immune Activation.

BACKGROUND: Inflammation is a key component of cardiac disease, with macrophages and T lymphocytes mediating essential roles in the progression to heart failure. Nonetheless, little insight exists on other immune subsets involved in the cardiotoxic response. METHODS: Here, we used single-cell RNA sequencing to map the cardiac immune composition in the standard murine nonischemic, pressure-overload heart failure model. By focusing our analysis on CD45+ cells, we obtained a higher resolution identification of the immune cell subsets in the heart, at early and late stages of disease and in controls. We then integrated our findings using multiparameter flow cytometry, immunohistochemistry, and tissue clarification immunofluorescence in mouse and human. RESULTS: We found that most major immune cell subpopulations, including macrophages, B cells, T cells and regulatory T cells, dendritic cells, Natural Killer cells, neutrophils, and mast cells are present in both healthy and diseased hearts. Most cell subsets are found within the myocardium, whereas mast cells are found also in the epicardium. Upon induction of pressure overload, immune activation occurs across the entire range of immune cell types. Activation led to upregulation of key subset-specific molecules, such as oncostatin M in proinflammatory macrophages and PD-1 in regulatory T cells, that may help explain clinical findings such as the refractivity of patients with heart failure to anti-tumor necrosis factor therapy and cardiac toxicity during anti-PD-1 cancer immunotherapy, respectively. CONCLUSIONS: Despite the absence of infectious agents or an autoimmune trigger, induction of disease leads to immune activation that involves far more cell types than previously thought, including neutrophils, B cells, Natural Killer cells, and mast cells. This opens up the field of cardioimmunology to further investigation by using toolkits that have already been developed to study the aforementioned immune subsets. The subset-specific molecules that mediate their activation may thus become useful targets for the diagnostics or therapy of heart failure.

Development, application and computational analysis of high-dimensional fluorescent antibody panels for single-cell flow cytometry.

The interrogation of single cells is revolutionizing biology, especially our understanding of the immune system. Flow cytometry is still one of the most versatile and high-throughput approaches for single-cell analysis, and its capability has been recently extended to detect up to 28 colors, thus approaching the utility of cytometry by time of flight (CyTOF). However, flow cytometry suffers from autofluorescence and spreading error (SE) generated by errors in the measurement of photons mainly at red and far-red wavelengths, which limit barcoding and the detection of dim markers. Consequently, development of 28-color fluorescent antibody panels for flow cytometry is laborious and time consuming. Here, we describe the steps that are required to successfully achieve 28-color measurement capability. To do this, we provide a reference map of the fluorescence spreading errors in the 28-color space to simplify panel design and predict the success of fluorescent antibody combinations. Finally, we provide detailed instructions for the computational analysis of such complex data by existing, popular algorithms (PhenoGraph and FlowSOM). We exemplify our approach by designing a high-dimensional panel to characterize the immune system, but we anticipate that our approach can be used to design any high-dimensional flow cytometry panel of choice. The full protocol takes a few days to complete, depending on the time spent on panel design and data analysis.

The Single-Cell Phenotypic Identity of Human CD8+ and CD4+ T Cells.

On antigen encounter, naïve CD8+ and CD4+ T cells differentiate into a large number of effector cells that migrate to inflamed tissues to fight infections or tumors. Following elimination of the target, a few cells remain in the long-term, the so-called memory T cells that are capable to reexpand and respond more vigorously on a second encounter with the cognate antigen. While the naïve T cell compartment is fairly homogenous, effector and memory T cells are largely diverse, comprising dozens of subsets with diverse functions, molecular characteristics, and localization in the body. In addition, CD4+ and, to some extent, also CD8+ T cells can differentiate into several effector subsets according to their pattern of cytokine expression, including the T helper 1 (Th1), Th2, and Th17 cells. It has become clear that specific subsets of T cells dominate different types of infections and pathological conditions and have different capacities to infiltrate and reject tumors. Their correct phenotypic identification is therefore of foremost importance for their live purification by magnetic or fluorescence-activated cell sorting and subsequent molecular characterization. Here, we present a comprehensive list of the main T cell subpopulations with a major focus on human cells along with their surface phenotypic properties.

Paths to expansion: Differential requirements of IRF4 in CD8+ T-cell expansion driven by antigen and homeostatic cytokines.

Interferon regulatory factor 4 (IRF4) regulates the clonal expansion and metabolic activity of activated T cells, but the precise context and mechanisms of its function in these processes are unclear. In this issue of the European Journal of Immunology, Miyakoda et al. [Eur. J. Immunol. 2018. 48: 1319-1328] show that IRF4 is required for activation and expansion of naïve and memory CD8+ T cells driven by T-cell receptor (TCR) signaling, but dispensable for memory CD8+ T-cell maintenance and homeostatic proliferation driven by homeostatic cytokines. The authors show that the function of IRF4 in CD8+ T-cell expansion is partially dependent upon activation of the PI3K/AKT pathway through direct or indirect attenuation of PTEN expression. These data shed light upon the differential intracellular pathways required for naïve and memory T cells to respond to self-antigens and/or homeostatic cytokines, and highlight the potential translational relevance of these findings in the context of immune reconstitution such as following allogeneic stem cell transplantation.

Background fluorescence and spreading error are major contributors of variability in high-dimensional flow cytometry data visualization by t-distributed stochastic neighboring embedding.

Multidimensional single-cell analysis requires approaches to visualize complex data in intuitive 2D graphs. In this regard, t-distributed stochastic neighboring embedding (tSNE) is the most popular algorithm for single-cell RNA sequencing and cytometry by time-of-flight (CyTOF), but its application to polychromatic flow cytometry, including the recently developed 30-parameter platform, is still under investigation. We identified differential distribution of background values between samples, generated by either background calculation or spreading error (SE), as a major source of variability in polychromatic flow cytometry data representation by tSNE, ultimately resulting in the identification of erroneous heterogeneity among cell populations. Biexponential transformation of raw data and limiting SE during panel development dramatically improved data visualization. These aspects must be taken into consideration when using computational approaches as discovery tools in large sets of samples from independent experiments or immunomonitoring in clinical trials.

High-dimensional single cell analysis identifies stem-like cytotoxic CD8+ T cells infiltrating human tumors.

CD8+ T cells infiltrating tumors are largely dysfunctional, but whether a subset maintains superior functionality remains ill defined. By high-dimensional single cell analysis of millions of CD8+ T cells from 53 individuals with lung cancer, we defined those subsets that are enriched in tumors compared with cancer-free tissues and blood. Besides exhausted and activated cells, we identified CXCR5+ TIM-3- CD8+ T cells with a partial exhausted phenotype, while retaining gene networks responsible for stem-like plasticity and cytotoxicity, as revealed by single cell sequencing of the whole transcriptome. Ex vivo, CXCR5+ TIM-3- CD8+ T cells displayed enhanced self-renewal and multipotency compared with more differentiated subsets and were largely polyfunctional. Analysis of inhibitory and costimulatory receptors revealed PD-1, TIGIT, and CD27 as possible targets of immunotherapy. We thus demonstrate a hierarchy of differentiation in the context of T cell exhaustion in human cancer similar to that of chronically infected mice, which is further shown to disappear with disease progression.

Vaccine antigens modulate the innate response of monocytes to Al(OH)3.

Aluminum-based adjuvants have widely been used in human vaccines since 1926. In the absence of antigens, aluminum-based adjuvants can initiate the inflammatory preparedness of innate cells, yet the impact of antigens on this response has not been investigated so far. In this study, we address the modulating effect of vaccine antigens on the monocyte-derived innate response by comparing processes initiated by Al(OH)3 and by Infanrix, an Al(OH)3-adjuvanted trivalent combination vaccine (DTaP), containing diphtheria toxoid (D), tetanus toxoid (T) and acellular pertussis (aP) vaccine antigens. A systems-wide analysis of stimulated monocytes was performed in which full proteome analysis was combined with targeted transcriptome analysis and cytokine analysis. This comprehensive study revealed four major differences in the monocyte response, between plain Al(OH)3 and DTaP stimulation conditions: (I) DTaP increased the anti-inflammatory cytokine IL-10, whereas Al(OH)3 did not; (II) Al(OH)3 increased the gene expression of IFNγ, IL-2 and IL-17a in contrast to the limited induction or even downregulation by DTaP; (III) increased expression of type I interferons-induced proteins was not observed upon DTaP stimulation, but was observed upon Al(OH)3 stimulation; (IV) opposing regulation of protein localization pathways was observed for Al(OH)3 and DTaP stimulation, related to the induction of exocytosis by Al(OH)3 alone. This study highlights that vaccine antigens can antagonize Al(OH)3-induced programming of the innate immune responses at the monocyte level.

Novel identified aluminum hydroxide-induced pathways prove monocyte activation and pro-inflammatory preparedness.

Aluminum-based adjuvants are the most widely used adjuvants in human vaccines. A comprehensive understanding of the mechanism of action of aluminum adjuvants at the molecular level, however, is still elusive. Here, we unravel the effects of aluminum hydroxide Al(OH)3 by a systems-wide analysis of the Al(OH)3-induced monocyte response. Cell response analysis by cytokine release was combined with (targeted) transcriptome and full proteome analysis. Results from this comprehensive study revealed two novel pathways to become activated upon monocyte stimulation with Al(OH)3: the first pathway was IFNß signaling possibly induced by DAMP sensing pathways like TLR or NOD1 activation, and second the HLA class I antigen processing and presentation pathway. Furthermore, known mechanisms of the adjuvant activity of Al(OH)3 were elucidated in more detail such as inflammasome and complement activation, homeostasis and HLA-class II upregulation, possibly related to increased IFNγ gene expression. Altogether, our study revealed which immunological pathways are activated upon stimulation of monocytes with Al(OH)3, refining our knowledge on the adjuvant effect of Al(OH)3 in primary monocytes. SIGNIFICANCE: Aluminum salts are the most used adjuvants in human vaccines but a comprehensive understanding of the working mechanism of alum adjuvants at the molecular level is still elusive. Our Systems Vaccinology approach, combining complementary molecular biological, immunological and mass spectrometry-based techniques gave a detailed insight in the molecular mechanisms and pathways induced by Al(OH)3 in primary monocytes. Several novel immunological relevant cellular pathways were identified: type I interferon secretion potentially induced by TLR and/or NOD like signaling, the activation of the inflammasome and the HLA Class-I and Class-II antigen presenting pathways induced by IFNγ. This study highlights the mechanisms of the most commonly used adjuvant in human vaccines by combing proteomics, transcriptomics and cytokine analysis revealing new potential mechanisms of action for Al(OH)3.

Bordetella pertussis outer membrane vesicle vaccine confers equal efficacy in mice with milder inflammatory responses compared to a whole-cell vaccine.

The demand for improved pertussis vaccines is urgent due to the resurgence of whooping cough. A deeper understanding of the mode of action of pertussis vaccines is required to achieve this improvement. The vaccine-induced effects of a candidate outer membrane vesicle vaccine (omvPV) and a classical protective but reactogenic whole cell vaccine (wPV) were comprehensively compared in mice. The comparison revealed essential qualitative and quantitative differences with respect to immunogenicity and adverse effects for these vaccines. Both vaccines stimulated a mixed systemic Th1/Th2/Th17 response. Remarkably, omvPV evoked higher IgG levels, lower systemic pro-inflammatory cytokine responses and enhanced splenic gene expression than wPV. The omvPV-induced transcriptome revealed gene signatures of the IFN-signaling pathway, anti-inflammatory signatures that attenuate LPS responses, anti-inflammatory metabolic signatures, and IgG responses. Upon intranasal challenge, both immunized groups were equally efficient in clearing Bordetella pertussis from the lungs. This study importantly shows that immunization with omvPV provides a milder inflammatory responses but with equal protection to bacterial colonization and induction of protective antibody and Th1/Th17 type immune responses compared to wPV. These results emphasize the potential of omvPV as a safe and effective next-generation pertussis vaccine.

Immunological Signatures after Bordetella pertussis Infection Demonstrate Importance of Pulmonary Innate Immune Cells.

Effective immunity against Bordetella pertussis is currently under discussion following the stacking evidence of pertussis resurgence in the vaccinated population. Natural immunity is more effective than vaccine-induced immunity indicating that knowledge on infection-induced responses may contribute to improve vaccination strategies. We applied a systems biology approach comprising microarray, flow cytometry and multiplex immunoassays to unravel the molecular and cellular signatures in unprotected mice and protected mice with infection-induced immunity, around a B. pertussis challenge. Pre-existing systemic memory Th1/Th17 cells, memory B-cells, and mucosal IgA specific for Ptx, Vag8, Fim2/3 were detected in the protected mice 56 days after an experimental infection. In addition, pre-existing high activity and reactivation of pulmonary innate cells such as alveolar macrophages, M-cells and goblet cells was detected. The pro-inflammatory responses in the lungs and serum, and neutrophil recruitment in the spleen upon an infectious challenge of unprotected mice were absent in protected mice. Instead, fast pulmonary immune responses in protected mice led to efficient bacterial clearance and harbored potential new gene markers that contribute to immunity against B. pertussis. These responses comprised of innate makers, such as Clca3, Retlna, Glycam1, Gp2, and Umod, next to adaptive markers, such as CCR6+ B-cells, CCR6+ Th17 cells and CXCR6+ T-cells as demonstrated by transcriptome analysis. In conclusion, besides effective Th1/Th17 and mucosal IgA responses, the primary infection-induced immunity benefits from activation of pulmonary resident innate immune cells, achieved by local pathogen-recognition. These molecular signatures of primary infection-induced immunity provided potential markers to improve vaccine-induced immunity against B. pertussis.