Azithromycin Clears Bordetella pertussis Infection in Mice but Also Modulates Innate and Adaptive Immune Responses and T Cell Memory

Treatment with the macrolide antibiotic azithromycin (AZM) is an important intervention for controlling infection of children with Bordetella pertussis and as a prophylaxis for preventing transmission to family members. However, antibiotics are known to have immunomodulatory effects independent of their antimicrobial activity. Here, we used a mouse model to examine the effects of AZM treatment on clearance of B. pertussis and induction of innate and adaptive immunity. We found that treatment of mice with AZM either 7 or 14 days post challenge effectively cleared the bacteria from the lungs. The numbers of innate immune cells in the lungs were significantly reduced in antibiotic-treated mice. Furthermore, AZM reduced the activation status of macrophages and dendritic cells, but only in mice treated on day 7. Early treatment with antibiotics also reduced the frequency of tissue-resident T cells and IL-17-producing cells in the lungs. To assess the immunomodulatory effects of AZM independent of its antimicrobial activity, mice were antibiotic treated during immunization with a whole cell pertussis (wP) vaccine. Protection against B. pertussis induced by immunization with wP was slightly reduced in AZM-treated mice. Antibiotic-treated wP-immunized mice had reduced numbers of lung-resident memory CD4 T cells and IL-17-production and reduced CD49d expression on splenic CD4 T cells after challenge, suggestive of impaired CD4 T cell memory. Taken together these results suggest that AZM can modulate the induction of memory CD4 T cells during B. pertussis infection, but this may in part be due to the clearance of B. pertussis and resulting loss of components that stimulate innate and adaptive immune response

Bystander activation of Bordetella pertussis-induced nasal tissue-resident memory CD4 T cells confers heterologous immunity to Klebsiella pneumoniae

Abstract Tissue-resident memory CD4 T ($T_{RM}$) cells induced by infection with Bordetella pertussis persist in respiratory tissues and confer long-term protective immunity against re-infection. However, it is not clear how they are maintained in respiratory tissues. Here we demonstrate that B. pertussis-specific CD4 $T_{RM}$ cells produce IL-17A in response to in vitro stimulation with LPS or heat-killed Klebsiella pneumoniae (HKKP) in the presence of dendritic cells. Furthermore, IL-17A-secreting CD4 $T_{RM}$ cells expand in the lung and nasal tissue of B. pertussis convalescent mice following in vivo administration of LPS or HKKP. Bystander activation of CD4 $T_{RM}$ cells was suppressed by anti-IL-12p40, but not by anti-MHCII antibodies. Furthermore, purified respiratory tissue-resident, but not circulating, CD4 T cells from convalescent mice produced IL-17A following direct stimulation with IL-23 and IL-1$\beta$ or IL-18. Intranasal immunization of mice with a whole cell pertussis vaccine induced respiratory CD4 $T_{RM}$ cells that were re-activated following stimulation with K. pneumoniae. Furthermore, the nasal pertussis vaccine conferred protective immunity against B. pertussis but also attenuated infection with K. pneumoniae. Our findings demonstrate CD4 $T_{RM}$ cells induced by respiratory infection or vaccination can undergo bystander activation and confer heterologous immunity to an unrelated respiratory pathogen

Hemmung der Oberflächenexpression des koinhibitorischen Rezeptors PD-1 auf tumorspezifischen T-Zellen mittels siRNA als möglicher klinischer Therapieansatz

Der koinhibitorische PD-1/PD-L1 Signaltransduktionsweg reguliert die T-Zellaktivität negativ und ist an der Aufrechterhaltung der peripheren Immuntoleranz beteiligt. Defekte führen im Tiermodell und im Menschen zu Autoimmunerkrankungen. In der Tumorimmunologie findet man den Rezeptor PD-1 auf tumorinfiltrierenden Lymphozyten, während der Ligand von zahlreichen Tumorarten exprimiert wird. Tumorspezifische T-Zellen können auf diesem Weg durch Tumoren gehemmt werden. Der PD-1/PD-L1 Signaltransduktionsweg stellt daher einen Ansatzpunkt für Immuntherapien dar. Eine Möglichkeit den Signaltransduktionsweg zu unterbrechen ist der systemische Einsatz blockierender Antikörper, wie sie für den koinhibitorischen Rezeptor CTLA-4 bereits in der Klinik verwendet werden. Der Einsatz von anti-CTLA-4 mAb ist jedoch mit Autoimmun-Nebenwirkungen verbunden, da die periphere Toleranz gebrochen wird. Um Autoimmun-Nebenwirkungen einer systemischen Blockade zu vermeiden, wurde RNAi durch retrovirale Transduktion mit siRNA-Vektoren als Methode gewählt, den Signaltransduktionsweg ausschließlich auf tumorspezifischen T-Zellen herunterzuregulieren. Im 2C TCR tg Mausmodell konnten durch Transduktion mit siRNA-Vektoren die Effektorfunktionen (Zytokinproduktion, Proliferation) der T-Zellen gegen PD-L1 exprimierende Tumorzellen verbessert werden. Im humanen System wurde der MART-spezifische 1D3 TCR, der in Kürze in einer klinischen Phase I Studie zum Einsatz kommen wird, verwendet, um TAA-spezifische T-Zellen zu generieren. Durch Doppeltransduktion der T-Zellen mit dem 1D3 TCR und einer gegen PD-1 gerichteten siRNA, wurden MART-spezifische T-Zellen mit PD-1 knock-down geschaffen. Die Expressionshemmung von PD-1 in 1D3 TCR transduzierten T-Zellen führte zu stärkerer Zytokinproduktion und lytischer Aktivität der T-Zellen in Gegenwart von PD-L1. Dieser Effekt ließ sich besonders ausgeprägt in CD4+ T-Zellen beobachten. Im Anschluss an die zur Zeit geplanten Studie zu retroviral eingebrachtem 1D3 TCR könnte in einer Folgestudie die Doppeltransduktion mit siRNA getestet werden, mit dem Ziel, die klinische Ansprechrate zu verbessern, ohne dabei die Autoimmunität, die durch nicht TAA-spezifische T-Zellen vermittelt wird zu verschärfen

NFAT signaling is indispensable for persistent memory responses of MCMV-specific CD8+ T cells.

Cytomegalovirus (CMV) induces a unique T cell response, where antigen-specific populations do not contract, but rather inflate during viral latency. It has been proposed that subclinical episodes of virus reactivation feed the inflation of CMV-specific memory cells by intermittently engaging T cell receptors (TCRs), but evidence of TCR engagement has remained lacking. Nuclear factor of activated T cells (NFAT) is a family of transcription factors, where NFATc1 and NFATc2 signal downstream of TCR in mature T lymphocytes. We show selective impacts of NFATc1 and/or NFATc2 genetic ablations on the long-term inflation of MCMV-specific CD8+ T cell responses despite largely maintained responses to acute infection. NFATc1 ablation elicited robust phenotypes in isolation, but the strongest effects were observed when both NFAT genes were missing. CMV control was impaired only when both NFATs were deleted in CD8+ T cells used in adoptive immunotherapy of immunodeficient mice. Transcriptome analyses revealed that T cell intrinsic NFAT is not necessary for CD8+ T cell priming, but rather for their maturation towards effector-memory and in particular the effector cells, which dominate the pool of inflationary cells

Immunization with whole cell but not acellular pertussis vaccines primes CD4 T <inf>RM</inf> cells that sustain protective immunity against nasal colonization with Bordetella pertussis

Protective immunity wanes rapidly after immunization of children with acellular pertussis (aP) vaccines and these vaccines do not prevent nasal colonization or transmission of Bordetella pertussis in baboons. In this study, we examined the role of tissue-resident memory T (TRM) cells in persistent protective immunity induced by infection or immunization with aP and whole-cell pertussis (wP) vaccines in mice. Immunization of mice with a wP vaccine protected against lung and nasal colonization, whereas an aP vaccine failed to protect in the nose. IL-17 and IFN-?-secreting CD69+CD4+ TRM cells were expanded in the lung and nasal tissue after B. pertussis challenge of mice immunized with wP, but not aP vaccines. However, previous infection induced the most persistent protection against nasal colonization and this correlated with potent induction of nasal tissue TRM cells, especially IL-17-secreting TRM cells. Blocking T cell migration to respiratory tissue during immunization with a wP vaccine impaired bacterial clearance, whereas transfer of TRM cells from convalescent or wP-immunized mice conferred protection to na?ve mice. Our findings reveal that previous infection or wP vaccination are significantly more effective than aP vaccination in conferring persistent protective immunity against B. pertussis and that this is mediated by respiratory TRM cell

image_1_Azithromycin Clears Bordetella pertussis Infection in Mice but Also Modulates Innate and Adaptive Immune Responses and T Cell Memory.jpeg

<p>Treatment with the macrolide antibiotic azithromycin (AZM) is an important intervention for controlling infection of children with Bordetella pertussis and as a prophylaxis for preventing transmission to family members. However, antibiotics are known to have immunomodulatory effects independent of their antimicrobial activity. Here, we used a mouse model to examine the effects of AZM treatment on clearance of B. pertussis and induction of innate and adaptive immunity. We found that treatment of mice with AZM either 7 or 14 days post challenge effectively cleared the bacteria from the lungs. The numbers of innate immune cells in the lungs were significantly reduced in antibiotic-treated mice. Furthermore, AZM reduced the activation status of macrophages and dendritic cells, but only in mice treated on day 7. Early treatment with antibiotics also reduced the frequency of tissue-resident T cells and IL-17-producing cells in the lungs. To assess the immunomodulatory effects of AZM independent of its antimicrobial activity, mice were antibiotic treated during immunization with a whole cell pertussis (wP) vaccine. Protection against B. pertussis induced by immunization with wP was slightly reduced in AZM-treated mice. Antibiotic-treated wP-immunized mice had reduced numbers of lung-resident memory CD4 T cells and IL-17-production and reduced CD49d expression on splenic CD4 T cells after challenge, suggestive of impaired CD4 T cell memory. Taken together these results suggest that AZM can modulate the induction of memory CD4 T cells during B. pertussis infection, but this may in part be due to the clearance of B. pertussis and resulting loss of components that stimulate innate and adaptive immune response.</p

CD8⁺ T cell differentiation status during chronic infection.

Mixed bone marrow chimeric animals were infected with 106 PFU of MCMV and sacrificed at 90 dpi. (A) Absolute count of KLRG1+CD27- CD8+ T cells in spleen and lungs of BMC animals at 90 dpi. (B) Frequency of CM and KLRG1-CD27+ T cells among primed CD45.2+/+ CD8+ T cells (CD44+CD11a+) from mesenteric LN of chronically infected mice. (C) Quantification of CM CD8+ T cells from CD45.2+/+ compartment in blood, spleen, lungs and mesenteric LN. (D) Absolute count of CX3CR1+ CD8+ T cells in spleen and lungs of BMC animals at 90 dpi. Data are pooled from two independent experiments and each dot represent one mouse. Statistically significant differences are highlighted; *, p (TIF)</p

NFAT signaling regulates effector CD8⁺ T cell differentiation during chronic infection.

Mixed BMC animals were infected with 106 PFU of MCMV and analyzed at 90 dpi. (A) Representative flow-cytometric plots showing KLRG1+CD27- and KLRG1-CD27+populations from BMC mice with WT and NFATc1c2 DKO BM. The plots are pre-gated on primed (CD44+CD11a+) CD45.2+ CD8+ T cells (live CD3+CD8+). (B) Pairwise comparison of KLRG1+CD27- and KLRG1-CD27+CD8+ T cell frequencies in blood, spleen and lungs of individual mice during chronic MCMV infection. Lines connect data from individual animals (C) Flow-cytometric plots showing representative central memory (CM) populations among primed blood CD8+ T cells of chronically infected mice (left). Kinetics of these CM populations in blood are shown on the right Lines connect group means at indicated time points, error bars are SEM. (D) Percentage of CM CD8+ T cells in spleen and lungs at 90 dpi. Bar plots represent the group average, error bars are SEM and each dot represents mouse. (E) Percentage of CM cells among M45 and M38 tetramer specific CD8+ T cells from spleen at 90 dpi. Bar plots represent mean ± SEM and each dot is a mouse. (F) Representative flow-cytometric plots of blood CD8+ T cells showing CXCR3+ population among primed (CD44+CD11a+) cells (left). Mean CXCR3+ CD8+ T cells population from blood, spleen and mesenteric LN at 90 dpi are shown as mean ± SEM, each dot is a mouse. (G) Representative flow-cytometric plots of blood CD8+ T cells showing CX3CR1+ population among primed (CD44+CD11a+) cells (left). Mean CX3CR1+ CD8+ T cells population from blood, spleen and lungs at 90 dpi are shown as mean ± SEM, each dot is a mouse. Data are pooled from two experiments and each dot represents one mouse, n≥7. Statistically significant differences are highlighted; *, p < 0.05; **, p < 0.01; ***, p < 0.001; (Mann-Whitney U Test); mean ± SEM values are plotted.</p

NFAT KO mice fail to mount inflationary CD8⁺ T cell response following chronic MCMV infection.

Animals lacking either NFATc1, NFATc2 or both were infected with 106 PFU of MCMV intraperitoneally. (A) Total CD8+ T cells and tetramer specific response kinetics in peripheral blood were tracked. Mean ± SEM are plotted for data pooled from 3 independent experiments (n≥12). Total CD4+ T cells, CD8+ T cells (B) and M38 tetramer+ T cell responses (C) in spleen at 90 dpi. (D) mice were sacrificed at 5 dpi to titrate the virus replication in spleen and lungs. (E) Infected mice were sacrificed 6 months post infection to quantify MCMV latent virus load, which is presented as virus copies per 105 host cells in spleen and salivary glands. Data in panel D and E are pooled from two experiments and each dot represents one mouse. Statistically significant differences are highlighted; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; (Mann-Whitney U Test).</p

Phenotype of inflationary CD8⁺ T cells during chronic infection.

Lymphocytes from spleen (A) or lungs (B) of mice infected for 3 months were stained for CD45.1, CD45.2 and CD8 expression as well as the indicated cell-surface molecules. The plots shown are gated on CD45.1+/-CD45.2+/- CD8+ T cells (shaded histogram) or CD45.1-/-CD45.2+/+ CD8+ T cells (black line) from the same sample. Data are representative of at least six individual mice per stain and two independent experiments. (TIF)</p