Human plasmacytoid dendritic cells acquire phagocytic capacity by TLR9 ligation in the presence of soluble factors produced by renal epithelial cells.

Plasmacytoid dendritic cells (pDCs) are antigen presenting cells specialized in viral recognition through Toll-like receptor (TLR)7 and TLR9, and produce vast amounts of interferon alpha upon ligation of these TLRs. We had previously demonstrated a strong influx of pDCs in the tubulointerstitium of renal biopsies at the time of acute rejection. However, the role of human pDCs in mediating acute or chronic allograft rejection remains elusive. pDCs are thought to have a limited capacity to ingest apoptotic cells, critical for inducing CD4+ T cell activation via indirect antigen presentation and subsequent activation of antibody producing B cells. Here we tested whether the function of pDCs is affected by their presence within the graft. Maturation and interferon alpha production by pDCs was enhanced when cells were activated in the presence of viable HK2 renal epithelial cells. Importantly, soluble factors produced by cytomegalovirus-infected (primary) epithelial or endothelial cells enhanced pDC activation and induced their capacity to phagocytose apoptotic cells. Phagocytosis was not induced by free virus or soluble factors from non-infected cells. Activated pDCs showed an enhanced CD4+ and CD8+ T cell allostimulatory capacity as well as a potent indirect alloantigen presentation. Granulocyte Macrophage-Colony Stimulating Factor is one of the soluble factors produced by renal epithelial cells that, combined with TLR9 ligation, induced this functional capacity. Thus, pDCs present in the rejecting allograft can contribute to alloimmunity and potentially act as important orchestrators in the manifestation of acute and chronic rejection.

The Breadth of Synthetic Long Peptide Vaccine-Induced CD8+ T Cell Responses Determines the Efficacy against Mouse Cytomegalovirus Infection.

There is an ultimate need for efficacious vaccines against human cytomegalovirus (HCMV), which causes severe morbidity and mortality among neonates and immunocompromised individuals. In this study we explored synthetic long peptide (SLP) vaccination as a platform modality to protect against mouse CMV (MCMV) infection in preclinical mouse models. In both C57BL/6 and BALB/c mouse strains, prime-booster vaccination with SLPs containing MHC class I restricted epitopes of MCMV resulted in the induction of strong and polyfunctional (i.e., IFN-γ+, TNF+, IL-2+) CD8+ T cell responses, equivalent in magnitude to those induced by the virus itself. SLP vaccination initially led to the formation of effector CD8+ T cells (KLRG1hi, CD44hi, CD127lo, CD62Llo), which eventually converted to a mixed central and effector-memory T cell phenotype. Markedly, the magnitude of the SLP vaccine-induced CD8+ T cell response was unrelated to the T cell functional avidity but correlated to the naive CD8+ T cell precursor frequency of each epitope. Vaccination with single SLPs displayed various levels of long-term protection against acute MCMV infection, but superior protection occurred after vaccination with a combination of SLPs. This finding underlines the importance of the breadth of the vaccine-induced CD8+ T cell response. Thus, SLP-based vaccines could be a potential strategy to prevent CMV-associated disease.

Viral Persistence Induces Antibody Inflation without Altering Antibody Avidity.

UNLABELLED: Antibodies are implicated in long-term immunity against numerous pathogens, and because of this property, antibody induction is the basis for many vaccines. Little is known about the influence of viral persistence on the evolving antibody response. Here, we examined the characteristics of antibody responses to persistent infection by employing the prototypic betaherpesvirus family member cytomegalovirus (CMV) in experimental mouse models. During the course of infection, mouse CMV (MCMV)-specific IgM and IgG responses are elicited; however, IgG levels gradually inflate in the persistent phase of infection while IgM levels are stably maintained. Whereas CD27-CD70 interactions are dispensable, the CD28/B7 costimulatory pathway is critical for the class switching of MCMV-specific IgM-to-IgG B cell responses, which corresponds to the CD28/B7-dependent formation of CD4(+)T follicular helper cells (TFH) and germinal center (GC) B cells. Furthermore, the initial viral inoculum dose dictates the height of the antibody levels during IgG antibody inflation and relates to the induction of long-lived plasma cells and memory B cells. Antibody avidity nonetheless is not altered after the establishment of viral persistence and occurs independently of the inoculum doses. However, repetitive challenge with intact viral particles, accompanied by increased GC reactivity, promotes the development of high-avidity IgG responses with neutralizing capacity. These insights can be used for the rational design of CMV-based vaccines aimed at inducing antibody responses. IMPORTANCE: Antibodies provide long-term protection to different pathogens. However, how antibody responses develop during persistent virus infection is not entirely clear. Here, we characterize factors that influence the virus-specific antibody response to persistent CMV. This study describes that during persistent infection, CMV-specific IgM antibody levels are stably maintained while IgG2b and IgG2c levels gradually inflate over time. In contrast, the IgG avidity remains similar after the establishment of viral persistence. The induction of T follicular helper cells and GC B cells requires CD4(+)T cell help and CD28/B7 costimulation signals and is essential for the development of CMV-specific IgG antibody responses. Furthermore, neutralizing CMV-specific antibodies appear to develop late after infection, yet the neutralizing capacity can be improved upon repetitive viral challenge that is associated with increased GC reactivity. The results described here could inform the use of CMV-based vaccines and may help to understand how our immune system copes with this persistent virus.

The Quantity of Autocrine IL-2 Governs the Expansion Potential of CD8+ T Cells.

Adequate responsiveness of CD8(+) T cell populations is of utmost importance for the efficacy of many vaccines and immunotherapeutic strategies against intracellular pathogens and cancer. In this study, we show in mouse models that the relative number of IL-2-producing cells within Ag-specific CD8(+) T cell populations predicts the population expansion capacity upon challenge. We further demonstrate that IL-2 producers constitute the best responding subset. Notably, we show that elevated production of IL-2 by CD8(+) T cells results in concomitant improved population expansion capacity and immunity. The amount of IL-2 produced on a per-cell basis essentially connects directly to the superior CD8(+) T cell population expansion. Together, our findings identified that autocrine IL-2 production operates in a dose-dependent fashion to facilitate the expansion potential of Ag-specific CD8(+) T cell populations, which may instigate ways to augment therapies depending on fit CD8(+) T cells.

Murine cytomegalovirus (CMV) infection via the intranasal route offers a robust model of immunity upon mucosal CMV infection.

Cytomegalovirus (CMV) is a ubiquitous virus, causing the most common congenital infection in humans, yet a vaccine against this virus is not available. Experimental studies of immunity against CMV in animal models of infection, such as the infection of mice with mouse CMV (MCMV), have relied mainly on parenteral infection protocols, although the virus naturally transmits by mucosal routes via body fluids. To characterize the biology of infections by mucosal routes, we compared the kinetics of virus replication, latent viral load and CD8 T-cell responses in lymphoid organs upon experimental intranasal (targeting the respiratory tract) and intragastric (targeting the digestive tract) infection with systemic intraperitoneal infection of two unrelated mouse strains. We observed that intranasal infection induced robust and long-term virus replication in the lungs and salivary glands but limited replication in the spleen. CD8 T-cell responses were somewhat weaker than upon intraperitoneal infection but showed similar kinetic profiles and phenotypes of antigen-specific cells. In contrast, intragastric infection resulted in abortive or poor virus replication in all tested organs and poor T-cell responses to the virus, especially at late times after infection. Consistent with the T-cell kinetics, the MCMV latent load was high in the lungs but low in the spleen of intranasally infected mice and lowest in all tested organs upon intragastric infection. In conclusion, we showed that intranasal but not intragastric infection of mice with MCMV represents a robust model to study the short- and long-term biology of CMV infection by a mucosal route.

Viral inoculum dose impacts memory T-cell inflation.

Memory T-cell inflation develops during certain persistent viral infections and is characterized by the accumulation and maintenance of large numbers of effector-memory T cells, albeit with varying degrees in size and phenotype among infected hosts. The underlying mechanisms that control memory T-cell inflation are not yet fully understood. Here, we dissected CMV-specific memory T-cell formation and its connection to the initial infectious dose by varying the inoculum size. After low dose inoculum with mouse CMV, the accumulation of inflationary memory T cells was severely hampered and correlated with reduced reservoirs of latent virus in nonhematopoietic cells and diminished antigen-driven T-cell proliferation. Moreover, lowering of the initial viral dose turned the characteristic effector memory-like inflationary T cells into more central memory-like cells as evidenced by the cell-surface phenotype of CD27(high) , CD62L(+) , CD127(+) , and KLRG1(-) , and by improved secondary expansion potential. These data show the impact of the viral inoculum on the degree of memory T-cell inflation and provide a rationale for the observed variation of human CMV-specific T-cell responses in terms of magnitude and phenotype.

Dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and T-cell activation.

The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T-cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre-)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte-derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4⁺ and CD8⁺ T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasome- and transporter associated with Ag processing-dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8⁺ T-cell activation.

CD27-CD70 costimulation controls T cell immunity during acute and persistent cytomegalovirus infection.

Cytomegaloviruses (CMVs) establish lifelong infections that are controlled in part by CD4(+) and CD8(+) T cells. To promote persistence, CMVs utilize multiple strategies to evade host immunity, including modulation of costimulatory molecules on infected antigen-presenting cells. In humans, CMV-specific memory T cells are characterized by the loss of CD27 expression, which suggests a critical role of the costimulatory receptor-ligand pair CD27-CD70 for the development of CMV-specific T cell immunity. In this study, the in vivo role of CD27-CD70 costimulation during mouse CMV infection was examined. During the acute phase of infection, the magnitudes of CMV-specific CD4(+) and CD8(+) T cell responses were decreased in mice with abrogated CD27-CD70 costimulation. Moreover, the accumulation of inflationary memory T cells during the persistent phase of infection and the ability to undergo secondary expansion required CD27-CD70 interactions. The downmodulation of CD27 expression, however, which occurs gradually and exclusively on inflationary memory T cells, is ligand independent. Furthermore, the IL-2 production in both noninflationary and inflationary CMV-specific T cells was dependent on CD27-CD70 costimulation. Collectively, these results highlight the importance of the CD27-CD70 costimulation pathway for the development of CMV-specific T cell immunity during acute and persistent infection.

Vaccine-induced effector-memory CD8+ T cell responses predict therapeutic efficacy against tumors.

CD8(+) T cells have the potential to attack and eradicate cancer cells. The efficacy of therapeutic vaccines against cancer, however, lacks defined immune correlates of tumor eradication after (therapeutic) vaccination based on features of Ag-specific T cell responses. In this study, we examined CD8(+) T cell responses elicited by various peptide and TLR agonist-based vaccine formulations in nontumor settings and show that the formation of CD62L(-)KLRG1(+) effector-memory CD8(+) T cells producing the effector cytokines IFN-γ and TNF predicts the degree of therapeutic efficacy of these vaccines against established s.c. tumors. Thus, characteristics of vaccine-induced CD8(+) T cell responses instill a predictive determinant for the efficacy of vaccines during tumor therapy.

Differential B7-CD28 costimulatory requirements for stable and inflationary mouse cytomegalovirus-specific memory CD8 T cell populations.

CMV establishes a lifelong persistent infection, and viral immune-modulating strategies are important in facilitating this. A particularly diverse CD8 T cell response develops as a result of this host-virus détente, with the CMV-specific memory T cell pool displaying unique functions and phenotypes. To gain insight into the factors that regulate CMV-specific CD8 T cell responses, we examined the influence of the B7-CD28 costimulatory pathway on magnitude, kinetics, and phenotype. Initial expansion of mouse CMV-specific CD8 T cells that establish stable memory pools was severely lower in mice lacking B7-CD28 signaling, and the resulting memory levels also remained reduced during persistent/latent infection. In contrast, expansion of CD8 T cells that undergo memory inflation during chronic infection was less affected in the absence of B7-CD28 costimulatory signals, eventually reaching the levels seen in wild-type mice at later times. Regardless of their differential requirements for B7-CD28 signals, both stable and inflationary memory T cell populations showed normal cytotoxic capacity. These results reveal that B7-CD28 costimulation differentially regulates the magnitude and kinetics of the multifaceted CD8 T cell response that develops during CMV infection.

OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum.

Immune checkpoint therapy (ICT) has the power to eradicate cancer, but the mechanisms that determine effective therapy-induced immune responses are not fully understood. Here, using high-dimensional single-cell profiling, we interrogate whether the landscape of T cell states in the peripheral blood predict responses to combinatorial targeting of the OX40 costimulatory and PD-1 inhibitory pathways. Single-cell RNA sequencing and mass cytometry expose systemic and dynamic activation states of therapy-responsive CD4+ and CD8+ T cells in tumor-bearing mice with expression of distinct natural killer (NK) cell receptors, granzymes, and chemokines/chemokine receptors. Moreover, similar NK cell receptor-expressing CD8+ T cells are also detected in the blood of immunotherapy-responsive cancer patients. Targeting the NK cell and chemokine receptors in tumor-bearing mice shows the functional importance of these receptors for therapy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use and targeting of dynamic biomarkers on T cells to improve cancer immunotherapy.

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

Dominant Antiviral CD8+ T Cell Responses Empower Prophylactic Antibody-Eliciting Vaccines Against Cytomegalovirus.

Human cytomegalovirus (HCMV) is an ubiquitous herpesvirus that can cause serious morbidity and mortality in immunocompromised or immune-immature individuals. A vaccine that induces immunity to CMV in these target populations is therefore highly needed. Previous attempts to generate efficacious CMV vaccines primarily focused on the induction of humoral immunity by eliciting neutralizing antibodies. Current insights encourage that a protective immune response to HCMV might benefit from the induction of virus-specific T cells. Whether addition of antiviral T cell responses enhances the protection by antibody-eliciting vaccines is however unclear. Here, we assessed this query in mouse CMV (MCMV) infection models by developing synthetic vaccines with humoral immunity potential, and deliberately adding antiviral CD8+ T cells. To induce antibodies against MCMV, we developed a DNA vaccine encoding either full-length, membrane bound glycoprotein B (gB) or a secreted variant lacking the transmembrane and intracellular domain (secreted (s)gB). Intradermal immunization with an increasing dose schedule of sgB and booster immunization provided robust viral-specific IgG responses and viral control. Combined vaccination of the sgB DNA vaccine with synthetic long peptides (SLP)-vaccines encoding MHC class I-restricted CMV epitopes, which elicit exclusively CD8+ T cell responses, significantly enhanced antiviral immunity. Thus, the combination of antibody and CD8+ T cell-eliciting vaccines provides a collaborative improvement of humoral and cellular immunity enabling enhanced protection against CMV.

Evaluation of immunological cross-reactivity between clade A9 high-risk human papillomavirus types on the basis of E6-Specific CD4+ memory T cell responses.

CD4(+) T cell responses against the E6 oncoprotein of human papillomavirus (HPV) type 16 and 5 closely related members of clade A9 (HPV31, 33, 35, 52, and 58) were charted in peripheral blood mononuclear cell cultures from healthy subjects and patients who underwent HPV16 E6/E7-specific vaccination. Initial analyses with overlapping peptide arrays showed that approximately one-half of the responding subjects displayed reactivity against corresponding E6 peptides from >or=2 HPV types. This suggested immunological cross-reactivity and complicated retrospective evaluation of the infection history of the healthy subjects. Importantly, further dissection of the response by means of enriched and clonal T cell cultures (with protein antigen instead of peptides) revealed that CD4(+) T cells that are capable of efficiently reacting against E6 antigen from multiple HPV types are rare and only occur when epitope sequences are highly conserved. Our data indicate that natural and vaccine-induced HPV16 E6-specific CD4(+) T cell responses are unlikely to mediate efficient cross-protection against other clade A9 members.

Cytomegalovirus infection and progressive differentiation of effector-memory T cells.

Primary cytomegalovirus (CMV) infection leads to strong innate and adaptive immune responses against the virus, which prevents serious disease. However, CMV infection can cause serious morbidity and mortality in individuals who are immunocompromised. The adaptive immune response to CMV is characterized by large populations of effector-memory (EM) T cells that are maintained lifelong, a process termed memory inflation. Recent findings indicate that infection with CMV leads to continuous differentiation of CMV-specific EM-like T cells and that high-dose infection accelerates this progression. Whether measures that counteract CMV infection, such as anti-viral drugs, targeting of latently infected cells, adoptive transfer of CMV-specific T cells, and vaccination strategies, are able to impact the progressive differentiation of CMV-specific EM-like cells is discussed.

A flexible MHC class I multimer loading system for large-scale detection of antigen-specific T cells.

Adaptive immunity is initiated by T cell recognition of specific antigens presented by major histocompatibility complexes (MHCs). MHC multimer technology has been developed for the detection, isolation, and characterization of T cells in infection, autoimmunity, and cancer. Here, we present a simple, fast, flexible, and efficient method to generate many different MHC class I (MHC I) multimers in parallel using temperature-mediated peptide exchange. We designed conditional peptides for HLA-A*02:01 and H-2Kb that form stable peptide-MHC I complexes at low temperatures, but dissociate when exposed to a defined elevated temperature. The resulting conditional MHC I complexes, either alone or prepared as ready-to-use multimers, can swiftly be loaded with peptides of choice without additional handling and within a short time frame. We demonstrate the ease and flexibility of this approach by monitoring the antiviral immune constitution in an allogeneic stem cell transplant recipient and by analyzing CD8+ T cell responses to viral epitopes in mice infected with lymphocytic choriomeningitis virus or cytomegalovirus.

FcγRI expression on macrophages is required for antibody-mediated tumor protection by cytomegalovirus-based vaccines.

Cytomegalovirus (CMV)-based vaccine vectors are promising vaccine platforms because they induce strong and long-lasting immune responses. Recently it has been shown that vaccination with a mouse CMV (MCMV) vector expressing the melanoma-specific antigen TRP2 (MCMV-TRP2) protects mice against outgrowth of TRP2-positive B16 melanoma tumors, and this protection was dependent on the induction of IgG antibodies. Here we demonstrate that, although mice lacking all receptors for the Fc part of IgG (FcγRs) develop normal IgG responses after MCMV-TRP2 vaccination, the protection against B16 melanoma was completely abrogated, indicating that FcγRs are indispensable in the downstream effector pathway of the polyclonal anti-TRP2 antibody response. By investigating compound FcγR-deficient mouse strains and by using immune cell type-specific cell ablation we show that the IgG antibody-mediated tumor protection elicited by MCMV-TRP2 mainly depends on FcγRI expression on macrophages, whereas FcγRIV plays only a modest role. Thus, tumor-specific antibody therapy might benefit from combination therapy that recruits FcγRI-expressing pro-inflammatory macrophages to the tumor micro-environment.

OX40 Stimulation Enhances Protective Immune Responses Induced After Vaccination With Attenuated Malaria Parasites.

Protection against a malaria infection can be achieved by immunization with live-attenuated Plasmodium sporozoites and while the precise mechanisms of protection remain unknown, T cell responses are thought to be critical in the elimination of infected liver cells. In cancer immunotherapies, agonistic antibodies that target T cell surface proteins, such as CD27, OX40 (CD134), and 4-1BB (CD137), have been used to enhance T cell function by increasing co-stimulation. In this study, we have analyzed the effect of agonistic OX40 monoclonal antibody treatment on protective immunity induced in mice immunized with genetically attenuated parasites (GAPs). OX40 stimulation enhanced protective immunity after vaccination as shown by an increase in the number of protected mice and delay to blood-stage infection after challenge with wild-type sporozoites. Consistent with the enhanced protective immunity enforced OX40 stimulation resulted in an increased expansion of antigen-experienced effector (CD11ahiCD44hi) CD8+ and CD4+ T cells in the liver and spleen and also increased IFN-γ and TNF producing CD4+ T cells in the liver and spleen. In addition, GAP immunization plus α-OX40 treatment significantly increased sporozoite-specific IgG responses. Thus, we demonstrate that targeting T cell costimulatory receptors can improve sporozoite-based vaccine efficacy.

Linking T cell epitopes to a common linear B cell epitope: A targeting and adjuvant strategy to improve T cell responses.

Immune complexes are potent mediators of cellular immunity and have been extensively studied for their disease mediating properties in humans and for their role in anti-cancer immunity. However, a viable approach to use antibody-complexed antigen as vehicle for specific immunotherapy has not yet reached clinical use. Since virtually all people have endogenous antibodies against tetanus toxoid (TTd), such commonly occurring antibodies are promising candidates to utilize for immune modulation. As an initial proof-of-concept we investigated if anti-tetanus IgG could induce potent cross-presentation of a conjugate with SIINFEKL, a MHC class I presented epitope of ovalbumin (OVA), to TTd. This protein conjugate enhanced OVA-specific CD8+ T cell responses when administrated to seropositive mice. Since TTd is poorly defined, we next investigated whether a synthetic peptide-peptide conjugate, with a chemically defined linear B cell epitope of tetanus toxin (TTx) origin, could improve cellular immune responses. Herein we identify one linear B cell epitope, here after named MTTE thru a screening of overlapping peptides from the alpha and beta region of TTx, and by assessment of the binding of pooled IgG, or individual human IgG from high-titer TTd vaccinated donors, to these peptides. Subsequently, we developed a chemical protocol to synthesize defined conjugates containing multiple copies of MTTE covalently attached to one or more T cell epitopes of choice. To demonstrate the potential of the above approach we showed that immune complexes of anti-MTTE antibodies with MTTE-containing conjugates are able to induce DC and T cell activation using model antigens.