Rama D. Yammani

Primate B-1 Cells Generate Antigen-Specific B Cell Responses to T Cell–Independent Type 2 Antigens

Ab responses to T cell–independent type 2 (TI-2) Ags, such as bacterial capsular polysaccharides, are critical for host defense. In mice, B-1b cells expressing a CD11b+FSChiCD21lo/−CD19hi phenotype play a key role in producing Abs against TI-2 Ags. In primates, a distinct IgM+CD27+ “memory” B cell population is thought to generate TI-2 Ab responses, and evidence for a B-1b–like cell population participating in these responses is lacking. In this article, we demonstrate that nonhuman primates (NHPs; African green monkeys and cynomolgus macaques) harbor serosal B cells expressing a CD11b+FSChiCD21lo/−CD80+/−CD19hi phenotype, constitutively active Stat3, and increased reactivity with phosphorylcholine, similar to murine peritoneal B-1a and B-1b cell populations. Like what is observed for murine B-1b cells, NHP CD11b+FSChiCD21lo/−CD19hi B cells dominate the Ag-specific B cell response and Ab production against the TI-2 Ag trinitrophenyl-Ficoll. Although Ag-specific IgM+ B cells expressing CD27 were not detected prior to immunization, Ag-specific CD11b+CD19hi B cells expressed and maintained an IgM+IgDloCD27+CD80+ phenotype following immunization. Thus, the murine and NHP B cell populations responding to trinitrophenyl-Ficoll are highly similar, with the main exception being that Ag-specific NHP B-1–like cells express CD27 following TI-2 Ag encounter. Therefore, murine B-1b and primate IgM+CD27+ “memory” B cell subsets proposed to produce TI-2 Ab responses may be highly related, if not identical. Overall, these data not only support that B-1–like cells are present in NHPs but also provide evidence that these cells perform the same functions attributed to murine B-1b cells.

Aging Promotes B-1b Cell Responses to Native, but Not Protein-Conjugated, Pneumococcal Polysaccharides: Implications for Vaccine Protection in Older Adults

The efficacy of different vaccines in protecting elderly individuals against Streptococcus pneumoniae infections is not clear. In the current study, aged mice (22-25 months old) exhibited significantly increased susceptibility to respiratory infection with serotype 3 S. pneumoniae relative to younger adult mice, regardless of whether mice were naive or immunized with native pneumococcal polysaccharide (PPS; Pneumovax23) or protein-PPS conjugate (Prevnar-13) vaccines. Nonetheless, Pneumovax-immunized aged mice developed limited bacteremia following respiratory challenge and exhibited significantly increased survival following systemic challenge relative to Prevnar-immune aged mice and young mice that had received either vaccine. This was explained by >10-fold increases in PPS-specific immunoglobulin G (IgG) levels in Pneumovax-immunized aged mice relative to other groups. Remarkably, PPS3-specific B-cell expansion, IgG switching, plasmablast differentiation, and spleen and bone marrow antibody-secreting cell frequencies were 10-fold higher in aged mice following Pneumovax immunization relative to young mice, due to significantly increased B-1b cell participation. In summary, this study highlights (1) the need to devise strategies to enhance respiratory immunity in aged populations, (2) the diverse responses young and aged populations generate to Pneumovax vs Prevnar vaccines, and (3) the potential value of exploiting B-1b cell responses in aged individuals for increased vaccine efficacy.

Regulation of maturation and activating potential in CD8+ versus CD8- dendritic cells following in vivo infection with vaccinia virus.

DC maturation is known to be a necessary step in the generation of an effective immune response. We have used vaccinia virus (VACV) as a model to investigate the regulation of DC subsets in vivo following infection. While a number of in vitro studies have shown that DC infected with VACV fail to undergo maturation, the effect of VACV infection on the maturation of and cytokine production by DC subsets in vivo remains less defined. We have found that following systemic infection with vaccinia virus, both CD8+ and CD8- dendritic cells are infected. The number of infected DC peaked at 6 h and was highly decreased by 24 h post-infection. In both subsets, there was evidence of generalized upregulation of costimulatory molecules. Surprisingly, this included vaccinia infected DC, suggesting the regulation of DC maturation in vivo is much more complex and likely influenced by DC extrinsic signals. However, while we observed generalized upregulation of costimulatory molecules, IL-12 production was restricted to a subset of non-infected cells in both the CD8+ and CD8- DC populations. Importantly, the control of IL-12 production was differentially dependent on MyD88 signaling. IL-12 production was ablated in the absence of MyD88 in CD8- DC, while it was unchanged in CD8+ DC. These findings provide new insights into the control of DC maturation in vivo and demonstrate that the regulation of maturation in vivo following virus infection can be differentially controlled in distinct types of DC.

PD-1 Suppresses Protective Immunity to Streptococcus pneumoniae through a B Cell–Intrinsic Mechanism

Despite the emergence of the programmed cell death 1 (PD-1):PD-1 ligand (PD-L) regulatory axis as a promising target for treating multiple human diseases, remarkably little is known about how this pathway regulates responses to extracellular bacterial infections. We found that PD-1−/− mice, as well as wild-type mice treated with a PD-1 blocking Ab, exhibited significantly increased survival against lethal Streptococcus pneumoniae infection following either priming with low-dose pneumococcal respiratory infection or S. pneumoniae-capsular polysaccharide immunization. Enhanced survival in mice with disrupted PD-1:PD-L interactions was explained by significantly increased proliferation, isotype switching, and IgG production by pneumococcal capsule-specific B cells. Both PD-L, B7-H1 and B7-DC, contributed to PD-1–mediated suppression of protective capsule-specific IgG. Importantly, PD-1 was induced on capsule-specific B cells and suppressed IgG production and protection against pneumococcal infection in a B cell–intrinsic manner. To our knowledge, these results provide the first demonstration of a physiologic role for B cell–intrinsic PD-1 expression in vivo. In summary, our study reveals that B cell–expressed PD-1 plays a central role in regulating protection against S. pneumoniae, and thereby represents a promising target for bolstering immunity to encapsulated bacteria.

CD8 marks a subpopulation of lung-derived dendritic cells with differential responsiveness to viral infection and toll-like receptor stimulation.

ABSTRACT An increasing number of studies suggest that individual subsets of dendritic cells (DC) exhibit distinct capabilities with regard to the generation of the adaptive immune response. In this study, we evaluated the properties of a relatively unexplored DC subset present in the lung-draining mediastinal lymph node. This subset expresses the airway dendritic cell marker CD103 together with CD8. These DC were of interest given that our previous studies using a model of respiratory infection with vaccinia virus revealed a distinct difference in the ability of CD103+ DC to prime T cells that correlated inversely with the expression of CD8, suggesting a differential role of these DC in the context of respiratory virus infection. To expand our understanding of the role of this DC population, we performed analyses to elucidate the phenotype, migratory capacity, responsiveness to innate stimuli, and priming capacity of CD8+ CD103+ DC. We found that expression of surface markers on these DC was similar to that of CD8− CD103+ DC, supporting their close relationship. Further, the two DC types were similar with regard to antigen uptake. However, although both CD103+ subsets originated from the lung, CD8-bearing CD103+ DC appeared in the lymph node with delayed kinetics following virus infection. While this subset exhibited increased responsiveness to a number of Toll-like receptor (TLR) agonists, their response to infection was virus specific, demonstrating poor responsiveness to vaccinia virus infection but robust maturation following infection with parainfluenza virus 5 or influenza virus. These findings show that CD8 marks a population of lung airway-derived DC with distinct migratory and maturation responses that likely contribute differentially to the immune response depending on the infecting pathogen.

Pivotal Advance: Nonfunctional lung effectors exhibit decreased calcium mobilization associated with reduced expression of ORAI1.

CD8+ T cells play a critical role in the clearance of respiratory pathogens. Thus, it is surprising that functional inactivation of lung effectors has been observed in many models of viral infection. Currently, the molecular defect responsible for the shut‐off of function in these cells is unknown. In the present study, we addressed this question using a model of respiratory infection with the paramyxovirus SV5. Nonfunctional cells were found to exhibit decreases in SOCE, resulting in reduced NFAT1 activation. Notably, function could be restored by the provision of increased levels of extracellular calcium. The reduced ability to mobilize calcium was associated with reduced expression of ORAI1, the CRAC channel subunit. These findings reveal a previously unknown mechanism for the negative regulation of function in effector T cells.

Changes in Functional but Not Structural Avidity during Differentiation of CD8+ Effector Cells In Vivo after Virus Infection

By the peak of the CD8+ T cell response, the effector cell pool consists of a heterogeneous population of cells that includes both those with an increased propensity to become long-lived memory cells (memory precursor effector cells; MPEC) and those that are terminally differentiated cells (short-lived effector cells; SLEC). Numerous studies have established the critical role that functional avidity plays in determining the in vivo efficacy of CD8+ effector cells. Currently, how functional avidity differs in MPEC versus SLEC and the evolution of this property within these two populations during the expansion and contraction of the response are unknown. The data presented in this study show that at the peak of the effector response generated after poxvirus infection, SLEC were of higher functional avidity than their MPEC counterpart. Over time, however, SLEC exhibited a decrease in peptide sensitivity. This is in contrast to MPEC, which showed a modest increase in peptide sensitivity as the response reached equilibrium. The decrease in functional avidity in SLEC was independent of CD8 modulation or the amount of Ag receptor expressed by the T cell. Instead, the loss in sensitivity was correlated with decreased expression and activation of ZAP70 and Lck, critical components of TCR membrane proximal signaling. These results highlight the potential contribution of avidity in the differentiation and evolution of the T cell effector response after viral infection.

High Viral Burden Restricts Short-Lived Effector Cell Number at Late Times Postinfection through Increased Natural Regulatory T Cell Expansion

Generating and maintaining a robust CD8+ T cell response in the face of high viral burden is vital for host survival. Further, balancing the differentiation of effectors along the memory precursor effector cell pathway versus the short-lived effector cell (SLEC) pathway may be critical in controlling the outcome of virus infection with regard to clearance and establishing protection. Although recent studies have identified several factors that have the capacity to regulate effector CD8+ T cell differentiation—for example, inflammatory cytokines—we are far from a complete understanding of how cells choose the memory precursor effector cell versus SLEC fate following infection. In this study, we have modulated the infectious dose of the poxvirus vaccinia virus as an approach to modulate the environment present during activation and expansion of virus-specific effector cells. Surprisingly, in the face of a high virus burden, the number of SLECs was decreased. This decrease was the result of increased natural regulatory T cells (Tregs) generated by high viral burden, as depletion of these cells restored SLECs. Our data suggest Treg modulation of differentiation occurs via competition for IL-2 during the late expansion period, as opposed to the time of T cell priming. These findings support a novel model wherein modulation of the Treg response as a result of high viral burden regulates late-stage SLEC number.

PD-L2 Regulates B-1 Cell Antibody Production against Phosphorylcholine through an IL-5–Dependent Mechanism

B-1 cells produce natural Abs which provide an integral first line of defense against pathogens while also performing important homeostatic housekeeping functions. In this study, we demonstrate that programmed cell death 1 ligand 2 (PD-L2) regulates the production of natural Abs against phosphorylcholine (PC). Naive PD-L2–deficient (PD-L2−/−) mice produced significantly more PC-reactive IgM and IgA. This afforded PD-L2−/− mice with selectively enhanced protection against PC-expressing nontypeable Haemophilus influenzae, but not PC-negative nontypeable Haemophilus influenzae, relative to wild-type mice. PD-L2−/− mice had significantly increased PC-specific CD138+ splenic plasmablasts bearing a B-1a phenotype, and produced PC-reactive Abs largely of the T15 Id. Importantly, PC-reactive B-1 cells expressed PD-L2 and irradiated chimeras demonstrated that B cell–intrinsic PD-L2 expression regulated PC-specific Ab production. In addition to increased PC-specific IgM, naive PD-L2−/− mice and irradiated chimeras reconstituted with PD-L2−/− B cells had significantly higher levels of IL-5, a potent stimulator of B-1 cell Ab production. PD-L2 mAb blockade of wild-type B-1 cells in culture significantly increased CD138 and Blimp1 expression and PC-specific IgM, but did not affect proliferation. PD-L2 mAb blockade significantly increased IL-5+ T cells in culture. Both IL-5 neutralization and STAT5 inhibition blunted the effects of PD-L2 mAb blockade on B-1 cells. Thus, B-1 cell–intrinsic PD-L2 expression inhibits IL-5 production by T cells and thereby limits natural Ab production by B-1 cells. These findings have broad implications for the development of therapeutic strategies aimed at altering natural Ab levels critical for protection against infectious disease, autoimmunity, allergy, cancer, and atherosclerosis.

Dose-dependent lymphocyte apoptosis following respiratory infection with Vaccinia virus.

Recently there has been renewed interest in poxvirus pathogenesis, especially with regard to infection via the respiratory route. Members of this family are known to produce a number of proteins that have the potential to negatively regulate the immune response. Vaccinia virus (VACV) has been used for a number of years as a model for the study of poxvirus infection. We have previously reported a dose-dependent decrease in virus-specific CD8(+) T cells following respiratory infection with VACV. In this study we have evaluated whether more generalized immunosuppressive effects are also observed following infection with a high dose of VACV. We have found that mice infected intranasally with a high, but non-lethal, dose of VACV exhibited significant weight loss as well as decreased thymocyte number. Although these mice mounted an immune response, there was a significant increase observed in bystander T and B cell apoptosis. While increased death was apparent in both naïve and activated/memory T cells populations, naïve T cells appeared more sensitive to this effect. These findings are important for our understanding of poxvirus regulation of the immune response and extends our previous understanding of VACV-mediated immunosuppression to include generalized apoptosis in the naïve and activated/memory repertoires.