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T regulatory cells (Tregs) play a critical role in controlling the immune response, often limiting pathogen-specific cells to curb immune-mediated damage. Studies in human infants have reported an increased representation of Tregs in these individuals. However, how these cells differ from those in adults at various sites and how they respond to activation signals is relatively unknown. In this study, we used a newborn nonhuman primate model to assess Treg populations present at multiple sites with regard to frequency and phenotype in comparison with those present in adult animals. We found that Foxp3+ cells were more highly represented in the T cell compartment of newborn nonhuman primates for all sites examined (i.e., the spleen, lung, and circulation). In the spleen and circulation, newborn-derived Tregs expressed significantly higher levels of Foxp3 and CD25 compared with adults, consistent with an effector phenotype. Strikingly, the phenotype of Tregs in the lungs of adult and infant animals was relatively similar, with both adult and newborn Tregs exhibiting a more uniform PD-1+CD39+ phenotype. Finally, in vitro, newborn Tregs exhibited an increased requirement for TCR engagement for survival. Further, these cells upregulated CD39 more robustly than their adult counterpart. Together, these data provide new insights into the quantity of Tregs in newborns, their activation state, and their potential to respond to activation signals.

Infection with influenza A virus can lead to increased susceptibility to subsequent bacterial infection, often with Streptococcus pneumoniae. Given the substantial modification of the lung environment that occurs following pathogen infection, there is significant potential for modulation of immune responses. In this study, we show that infection of mice with influenza virus, followed by the noninvasive EF3030 strain of Streptococcus pneumoniae, leads to a significant decrease in the virus-specific CD8+ T cell response in the lung. Adoptive-transfer studies suggest that this reduction contributes to disease in coinfected animals. The reduced number of lung effector cells in coinfected animals was associated with increased death, as well as a reduction in cytokine production in surviving cells. Further, cells that retained the ability to produce IFN-γ exhibited a decreased potential for coproduction of TNF-α. Reduced cytokine production was directly correlated with a decrease in the level of mRNA. Negative regulation of cells in the mediastinal lymph node was minimal compared with that present in the lung, supporting a model of selective regulation in the tissue harboring high pathogen burden. These results show that entry of a coinfecting pathogen can have profound immunoregulatory effects on an ongoing immune response. Together, these findings reveal a novel dynamic interplay between concurrently infecting pathogens and the adaptive immune system.

Generation of effective antibody responses following virus infection is challenging in neonates as a result of impairments in the innate and adaptive immune systems that characterize early life. This leaves them particularly susceptible to severe disease following virus infection, e.g. influenza A virus. A potential contributor to the efficacy of the immune response generated following infection is the epitope specificity of the elicited antibodies. Previous studies in adult mice have reported a defined and reproducible pattern of immunodominance among antibodies directed to the neutralizing epitopes on the head of the influenza hemagglutinin (HA) molecule. The impact of age on the immunodominance pattern of HA-specific antibodies has not been explored. To address this, epitope recognition was quantified in plasma collected from newborn and adult African Green monkeys on d14 following PR8 influenza virus infection. For these analyses we used HA molecules engineered to singly express each of the neutralizing epitopes identified on the HA head. In addition, we used a construct that restricts recognition to the stem region of PR8 HA. Our analyses revealed a similar pattern of recognition by HA-specific IgG between infants and adults in the 14 day period following infection. In contrast, the HA-specific IgM pools exhibited distinct binding patterns in these two groups. The most striking of these differences was in the production of antibodies capable of recognizing the stem portion of HA. These data suggest antibody immunodominance patterns may be modulated with age and sheds new light on the regulation of potentially broadly protective stem responses to influenza virus.

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.

CTL that possess a high functional avidity are known to be optimal for the clearance of pathogens in vivo. We have shown that the amount of peptide encountered by a CD8+ CTL determines its functional avidity. Notably, in these studies nonprofessional APC were used. However, it is mature dendritic cells (DC) that are predominantly responsible for the activation of naive T cells in vivo. Whether DC also direct dose dependent-differences in avidity is unknown. In this work we examined the ability of mature DC presenting a high vs low level of peptide to generate CTL of distinct avidities. In contrast to what was observed with nonprofessional APC, CTL generated by stimulation with mature DC were of high avidity regardless of the amount of peptide presented. This DC property may promote generation of highly effective CTL that retain plasticity, which would allow the tuning of avidity in the periphery to promote optimal pathogen recognition and clearance.

CD80 and CD86 have been shown to play a critical role in the optimal activation of T cells. Although these two molecules bind the same ligand, CD28, the question of whether CD80 and CD86 provide unique signals or serve redundant roles remains controversial. Previous studies have suggested that CD80 binding to CD28 may be superior to CD86 for the activation of naive CD8+ T cells. This study provides a potential mechanism to explain these observations. Our study demonstrates a previously unappreciated role for CD80, its superiority over CD86 in promoting CD25 expression, increasing both the number of cells that express CD25 and the level expressed on a per cell basis. These findings provide new insights into the role of CD80 vs CD86 and have important implications for the design of vaccines and immunotherapeutics aimed at the generation of a robust CD8+ T cell response in vivo.

Previously, we observed that high-avidity CTL are much more effective in vivo than low-avidity CTL in elimination of infected cells, but the mechanisms behind their superior activity remained unclear. In this study, we identify two complementary mechanisms: 1) high-avidity CTL lyse infected cells earlier in the course of a viral infection by recognizing lower Ag densities than those distinguished by low-avidity CTL and 2) they initiate lysis of target cells more rapidly at any given Ag density. Alternative mechanisms were excluded, including: 1) the possibility that low-avidity CTL might control virus given more time (virus levels remained as high at 6 days following transfer as at 3 days) and 2) that differences in efficacy might be correlated with homing ability. Furthermore, adoptive transfer of high- and low-avidity CTL into SCID mice demonstrated that transfer of a 10-fold greater amount of low-avidity CTL could only partially compensate for their decreased ability to eliminate infected cells. Thus, we conclude that high-avidity CTL exploit two complementary mechanisms that combine to prevent the spread of virus within the animal: earlier recognition of infected cells when little viral protein has been made and more rapid lysis of infected cells.

During an immune response, CTL clones are present that exhibit a wide spectrum of peptide sensitivities. While the property of functional avidity has been well documented, the range of mechanism(s) by which this T cell property can be regulated is unknown. For example, an unresolved question is whether distinct naïve T cell subsets exist that display “inherent” differences in their peptide requirements for activation, i.e. functional avidity. In this scenario distinct naïve T cell clones would selectively respond upon encountering a high vs. low antigen concentration. Alternatively, differences in peptide sensitivity could be an “induced” property within the responding CTL population following encounter with an APC. Although the latter model is consistent with data from previous studies, formal demonstration that a single clone can give rise to both high and low avidity daughter cells is lacking. Using a TCR transgenic model in which both high and low avidity CTL can be generated, we now demonstrate that naïve T cells do not exhibit differences in the amount of peptide required for conjugate formation and subsequent proliferation/activation. Furthermore, we show that individual T cell clones are capable of differentiating into either a high or low avidity CTL. These results provide the first formal demonstration that an individual cell can give rise to both high and low avidity progeny, suggesting that avidity modulation at the level of an individual cell may be an important contributor to the functional avidity of the CD8+ T cell response generated in vivo. This work was supported by National Institutes of Health grant R01 AI43591 to M.A.A-M.

CTLs that display high functional avidity are known to be optimal for pathogen clearance. However, the molecular mechanism that allows high avidity CTLs to respond at very low levels of peptide antigen remains to be elucidated. To investigate this question, we compared TCR signal transduction events in high and low avidity CD8+ T-cells generated from OT-I TCR transgenic mice. Upon stimulation with APCs preloaded with a range of Ova257-264 peptide concentrations (10^-6, 10^-9 and 10^-12 M), we found that high avidity CD8+ T-cells induced higher levels of MAPK ERK-1/2 phosphorylation as well as higher levels of the dephosphorylated form of NFAT at each peptide concentration used for stimulation. We next determined whether the observed divergence in NFAT activation was reflected in differences in calcium mobilization. We found efficient mobilization of Ca2+ in high avidity cells even when the lowest concentration of peptide was used for stimulation, a level of peptide at which no increase in calcium was observed in low avidity cells. The decreased peptide requirement for calcium mobilization in high avidity cells correlated with a decreased peptide requirement for ZAP-70 activation and CD3ζ phosphorylation, the latter of which was higher and prolonged in high versus low avidity cells. Thus the functional avidity of the CD8+ effector cells studies here is controlled by differential regulation of the most membrane proximal event, phosphorylation of CD3ζ. Studies are currently underway to determine the mechanism by which high avidity cells acheive CD3ζ phosphorylation at limiting peptide levels.

Recently there has been renewed interest in poxvirus pathogenesis. Vaccinia virus (VACV) serves as a useful model for the study of poxvirus infection in vivo. Understanding how vaccinia virus generates immunity necessitates an appreciation for how this virus interacts with dendritic cells (DC), which are the most potent activators of naïve T cells. A number of in vitro studies have shown that DC infected with VACV fail to undergo maturation. However, the effect of VACV infection on DC in vivo remains less defined. We have found that following intravenous administration of vaccinia virus, both CD8+ and CD8− dendritic cells are infected. The number of infected DC peaks at 6 hours and is highly decreased by 24h postinfection. Dendritic cells infected with vaccinia virus have an immature phenotype. However, non-infected dendritic cells, both CD8+ and CD8− subsets, show increased expression of CD80, CD86, and CD40, suggesting that there is a generalized maturation of non-infected DC. In contrast to the generalized upregulation of costimulatory molecules, cytokine analysis suggests that only a subset of these mature DC may be relevant for activation of T cells, as IL-12 production is restricted to a small percentage of cells. These findings provide new insights into the control of DC maturation in vivo following vaccinia virus infection. This work was supported by NIH grant P01 AI60642.