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.

Nasal vaccines for pertussis.

Whooping cough, caused by Bordetella pertussis, is still a major cause of morbidity and mortality worldwide. Current acellular pertussis (aP) vaccines induce potent circulating IgG and prevent severe disease in children/adults and in infants born to vaccinated mothers. However, they do not prevent nasal infection, allowing asymptomatic transmission of B. pertussis. Studies in animal models have demonstrated that, unlike natural infection, immunization with aP vaccines fails to induce secretory immunoglobulin A (IgA) or interleukin-17 (IL-17)-secreting tissue-resident memory CD4 T (TRM) cells, required for sustained sterilizing immunity in the nasal mucosa. Live-attenuated vaccines or aP vaccines formulated with novel adjuvants that induce respiratory IgA and TRM cells, especially when delivered by the nasal route, are in development and have considerable promise as next-generation vaccines against pertussis.

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

Tissue-resident memory CD4 T (TRM ) cells induced by infection with Bordetella pertussis persist in respiratory tissues and confer long-term protective immunity against reinfection. However, it is not clear how they are maintained in respiratory tissues. Here, we demonstrate that B. pertussis-specific CD4 TRM 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 TRM 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 TRM 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-1ß or IL-18. Intranasal immunization of mice with a whole-cell pertussis vaccine induced respiratory CD4 TRM cells that were reactivated 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 that CD4 TRM cells induced by respiratory infection or vaccination can undergo bystander activation and confer heterologous immunity to an unrelated respiratory pathogen.

Turnover of Murine Cytomegalovirus-Expanded CD8+ T Cells Is Similar to That of Memory Phenotype T Cells and Independent of the Magnitude of the Response.

The potential of memory T cells to provide protection against reinfection is beyond question. Yet, it remains debated whether long-term T cell memory is due to long-lived memory cells. There is ample evidence that blood-derived memory phenotype CD8+ T cells maintain themselves through cell division, rather than through longevity of individual cells. It has recently been proposed, however, that there may be heterogeneity in the lifespans of memory T cells, depending on factors such as exposure to cognate Ag. CMV infection induces not only conventional, contracting T cell responses, but also inflationary CD8+ T cell responses, which are maintained at unusually high numbers, and are even thought to continue to expand over time. It has been proposed that such inflating T cell responses result from the accumulation of relatively long-lived CMV-specific memory CD8+ T cells. Using in vivo deuterium labeling and mathematical modeling, we found that the average production rates and expected lifespans of mouse CMV-specific CD8+ T cells are very similar to those of bulk memory-phenotype CD8+ T cells. Even CMV-specific inflationary CD8+ T cell responses that differ 3-fold in size were found to turn over at similar rates.

IL-17 mediates protective immunity against nasal infection with Bordetella pertussis by mobilizing neutrophils, especially Siglec-F+ neutrophils.

Understanding the mechanism of protective immunity in the nasal mucosae is central to the design of more effective vaccines that prevent nasal infection and transmission of Bordetella pertussis. We found significant infiltration of IL-17-secreting CD4+ tissue-resident memory T (TRM) cells and Siglec-F+ neutrophils into the nasal tissue during primary infection with B. pertussis. Il17A-/- mice had significantly higher bacterial load in the nasal mucosae, associated with significantly reduced infiltration of Siglec-F+ neutrophils. Re-infected convalescent mice rapidly cleared B. pertussis from the nasal cavity and this was associated with local expansion of IL-17-producing CD4+ TRM cells. Depletion of CD4 T cells from the nasal tissue during primary infection or after re-challenge of convalescent mice significantly delayed clearance of bacteria from the nasal mucosae. Protection was lost in Il17A-/- mice and this was associated with significantly less infiltration of Siglec-F+ neutrophils and antimicrobial peptide (AMP) production. Finally, depletion of neutrophils reduced the clearance of B. pertussis following re-challenge of convalescent mice. Our findings demonstrate that IL-17 plays a critical role in natural and acquired immunity to B. pertussis in the nasal mucosae and this effect is mediated by mobilizing neutrophils, especially Siglec-F+ neutrophils, which have high neutrophil extracellular trap (NET) activity.

Reverse TCR repertoire evolution toward dominant low-affinity clones during chronic CMV infection.

Adaptive evolution is a key feature of T cell immunity. During acute immune responses, T cells harboring high-affinity T cell antigen receptors (TCRs) are preferentially expanded, but whether affinity maturation by clonal selection continues through the course of chronic infections remains unresolved. Here we investigated the evolution of the TCR repertoire and its affinity during the course of infection with cytomegalovirus, which elicits large T cell populations in humans and mice. Using single-cell and bulk TCR sequencing and structural affinity analyses of cytomegalovirus-specific T cells, and through the generation and in vivo monitoring of defined TCR repertoires, we found that the immunodominance of high-affinity T cell clones declined during the chronic infection phase, likely due to cellular senescence. These data showed that under conditions of chronic antigen exposure, low-affinity TCRs preferentially expanded within the TCR repertoire, with implications for immunotherapeutic strategies.

Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge.

Cytomegalovirus (CMV) is a ubiquitous ß-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.

Immunization with whole cell but not acellular pertussis vaccines primes CD4 TRM 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-γ-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 naïve 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 cells.

Sustained protective immunity against Bordetella pertussis nasal colonization by intranasal immunization with a vaccine-adjuvant combination that induces IL-17-secreting TRM cells.

Current acellular pertussis (aP) vaccines induce strong antibody and Th2 responses but fail to protect against nasal colonization and transmission of Bordetella pertussis. Furthermore, immunity wanes rapidly after immunization. We have developed a novel adjuvant combination (called LP-GMP), comprising c-di-GMP, an intracellular receptor stimulator of interferon genes (STING) agonist, and LP1569, a TLR2 agonist from B. pertussis, which synergistically induces production of IFN-ß, IL-12 and IL-23, and maturation of dendritic cells. Parenteral immunization of mice with an experimental aP vaccine formulated with LP-GMP promoted Th1 and Th17 responses and conferred protection against lung infection with B. pertussis. Intranasal immunization with the same aP vaccine-induced potent B. pertussis-specific Th17 responses and IL-17-secreting respiratory tissue-resident memory (TRM) CD4 T cells, and conferred a high level of protection against nasal colonization as well as lung infection, which was sustained for at least 10 months. Furthermore, long-term protection against nasal colonization with B. pertussis correlated with the number of IL-17-secreting TRM cells in nasal tissue. Our study has identified an approach for inducing IL-17-secreting TRM cells that sustain sterilizing immunity against nasal colonization of mice with B. pertussis, and could form the basis of a third generation pertussis vaccine for humans.

IL-17-dependent SIgA-mediated protection against nasal Bordetella pertussis infection by live attenuated BPZE1 vaccine.

BPZE1 is a live attenuated Bordetella pertussis vaccine for nasal administration to mimic the natural route of infection. Here, we studied the mechanism of BPZE1-induced immunity in the murine nasal cavity in contrast to acellular vaccine (aPV), although both vaccines protected against lung colonization. Transfer of splenocytes or serum from BPZE1-vaccinated or aPV-vaccinated mice protected naïve mice against lung colonization but not against nasal colonization. However, transfer of nasal washes from BPZE1-vaccinated mice resulted in protection against nasal colonization, which was lost in IgA-deficient or poly-Ig receptor-deficient mice, indicating that it depends on secretory IgA (SIgA) induction induced in the nose. BPZE1-induced protection against nasal colonization was long-lived despite the relatively rapid decay of SIgA, indicating a potent BPZE1-induced local memory response, likely due to CD4+ tissue-resident memory T cells induced in the nose by BPZE1. These cells produced interleukin-17 (IL-17), known to be important for SIgA secretion. Furthermore, BPZE1 failed to protect Il17-/- mice against nasal colonization by B. pertussis and induced only background levels of nasal SIgA. Thus, our results show important differences in the protective mechanism between the upper and the lower murine respiratory tract and demonstrate an IL-17-dependent SIgA-mediated mechanism of BPZE1-induced protection against B. pertussis nasopharyngeal colonization.

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.

Immune Protection by a Cytomegalovirus Vaccine Vector Expressing a Single Low-Avidity Epitope.

Experimental CMV-based vaccine vectors expressing a single MHC class I-restricted high-avidity epitope provided strong, T cell-dependent protection against viruses or tumors. In this study we tested the low-avidity epitope KCSRNRQYL, and show that a mouse CMV (MCMV) vector provides complete immune control of recombinant vaccinia virus expressing the same epitope if KCSRNRQYL is expressed within the immediate-early MCMV gene ie2 The same epitope expressed within the early M45 gene provided no protection, although MCMV vectors expressing the high-avidity epitope SSIEFARL induced protective immunity irrespective of gene expression context. Immune protection was matched by Ag-induced, long-term expansion of effector memory CD8 T cells, regardless of epitope avidity. We explained this pattern by observing regularities in Ag competition, where responses to high-avidity epitopes outcompeted weaker ones expressed later in the replicative cycle of the virus. Conversely, robust and early expression of a low-avidity epitope compensated its weak intrinsic antigenicity, resulting in strong and sustained immunity and immune protection.

UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene.

Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 (also known as vICA), binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called M36, and its protein product (pM36) is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMVUL36 inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo.

Peptide Processing Is Critical for T-Cell Memory Inflation and May Be Optimized to Improve Immune Protection by CMV-Based Vaccine Vectors.

Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy.

Conventional Dendritic Cells Confer Protection against Mouse Cytomegalovirus Infection via TLR9 and MyD88 Signaling.

Cytomegalovirus (CMV) is an opportunistic virus severely infecting immunocompromised individuals. In mice, endosomal Toll-like receptor 9 (TLR9) and downstream myeloid differentiation factor 88 (MyD88) are central to activating innate immune responses against mouse CMV (MCMV). In this respect, the cell-specific contribution of these pathways in initiating anti-MCMV immunity remains unclear. Using transgenic mice, we demonstrate that TLR9/MyD88 signaling selectively in CD11c+ dendritic cells (DCs) strongly enhances MCMV clearance by boosting natural killer (NK) cell CD69 expression and IFN-γ production. In addition, we show that in the absence of plasmacytoid DCs (pDCs), conventional DCs (cDCs) promote robust NK cell effector function and MCMV clearance in a TLR9/MyD88-dependent manner. Simultaneously, cDC-derived IL-15 regulates NK cell degranulation by TLR9/MyD88-independent mechanisms. Overall, we compartmentalize the cellular contribution of TLR9 and MyD88 signaling in individual DC subsets and evaluate the mechanism by which cDCs control MCMV immunity.

Immune Protection against Virus Challenge in Aging Mice Is Not Affected by Latent Herpesviral Infections.

Latent herpesvirus infections alter immune homeostasis. To understand if this results in aging-related loss of immune protection against emerging infections, we challenged old mice carrying latent mouse cytomegalovirus (CMV), herpes simplex virus 1 (HSV-1), and/or murine gammaherpesvirus 68 (MHV-68) with influenza virus, West Nile virus (WNV), or vesicular stomatitis virus (VSV). We observed no increase in mortality or weight loss compared to results seen with herpesvirus-negative counterparts and a relative but not absolute reduction in CD8 responses to acute infections. Therefore, the presence of herpesviruses does not appear to increase susceptibility to emerging infections in aging patients.

Osteopontin deficiency affects imiquimod-induced psoriasis-like murine skin inflammation and lymphocyte distribution in skin, draining lymph nodes and spleen.

Osteopontin (OPN) that enhances autoimmunity is expressed in psoriasis lesions; however, its functions in psoriatic inflammation are unknown. We investigated the role of OPN in OPN deficient mice (OPN-/-) by inducing psoriasis-like inflammation through skin application of imiquimod (IMQ). OPN-/- mice treated with IMQ showed delayed onset ear swelling and attracted less inflammatory cells to the skin. IMQ-induced lymph node swelling was reduced in the absence of OPN, and IMQ-mediated expansion of B cells was inhibited. Further, reduction of CD4(+) T-cell numbers by IMQ in lymph nodes was suppressed in OPN-/- mice, with an increase in the CD4/CD8 ratio. A comparable pattern was found in spleen. Importantly, IMQ-induced IL-17 and IL-4 expression by CD4(+) lymph node T cells was reduced in OPN-/- mice. In conclusion, OPN may modulate psoriasis-like inflammation through altering lymphocyte distribution in skin and draining lymph nodes and by inducing IL-17 expression of inflammatory T cells.

Reduced CD18 levels drive regulatory T cell conversion into Th17 cells in the CD18hypo PL/J mouse model of psoriasis.

Defective development and function of CD4(+)CD25(high+)Foxp3(+) regulatory T cells (Tregs) contribute to the pathogenesis of psoriasis and other autoimmune diseases. Little is known about the influence of adhesions molecules on the differentiation of Foxp3(+) Tregs into proinflammatory Th17 cells occurring in lesional skin and blood of psoriasis patients. In the CD18(hypo) PL/J mouse model of psoriasis, reduced expression of CD18/ß2 integrin to 2-16% of wild-type levels is associated with progressive loss of Tregs, impaired cell-cell contact between Tregs and dendritic cells (DCs), as well as Treg dysfunction as reported earlier. In the present investigation, Tregs derived from CD18(hypo) PL/J mice were analyzed for their propensity to differentiate into IL-17-producing Th17 cells in vivo and in in vitro Treg-DC cocultures. Adoptively transferred CD18(hypo) PL/J Tregs were more inclined toward conversion into IL-17-producing Th17 cells in vivo in an inflammatory as well as noninflammatory environment compared with CD18(wt) PL/J Tregs. Addition of neutralizing Ab against CD18 to Treg-DC cocultures in vitro promoted conversion of CD18(wt) PL/J Tregs to Th17 cells in a dose-dependent manner similar to conversion rates of CD18(hypo) PL/J Tregs. Reduced thymic output of naturally occurring Tregs and peripheral conversion of Tregs into Th17 cells therefore both contribute to the loss of Tregs and the psoriasiform dermatitis observed in CD18(hypo) PL/J mice. Our data overall indicate that CD18 expression levels impact Treg development as well as Treg plasticity and that differentiation of Tregs into IL-17-producing Th17 cells is distinctly facilitated by a subtotal deficiency of CD18.

Reduced tumor-antigen density leads to PD-1/PD-L1-mediated impairment of partially exhausted CD8⁺ T cells.

Clinical progression of cancer patients is often observed despite the presence of tumor-reactive T cells. Co-inhibitory ligands of the B7 superfamily have been postulated to play a part in this tumor-immune escape. One of these molecules, PD-L1 (B7-H1, CD274), is widely expressed on tumor cells and has been shown to mediate T-cell inhibition. However, attempts to correlate PD-L1 tumor expression with negative prognosis have been conflicting. To better understand when PD-1/PD-L1-mediated inhibition contributes to the functional impairment of tumor-specific CD8(+) T cells, we varied the levels of antigen density and/or PD-L1 expression at the surface of tumor cells and exposed them to CD8(+) T cells at different levels of functional exhaustion. We found that the gradual reduction of cognate antigen expression by PD-L1-expressing tumor cells increased the susceptibility of partially exhausted T cells to PD-1/PD-L1-mediated inhibition in vitro as well as in vivo. In conclusion, chronically stimulated CD8(+) T cells become sensitive to PD-1/PD-L1-mediated functional inhibition upon low antigen detection; a setting which is likely involved during tumor-immune escape.