Tonic B cell antigen receptor signals supply an NF-kappaB substrate for prosurvival BLyS signaling.

The survival of transitional and mature B cells requires both the B cell antigen receptor (BCR) and BLyS receptor 3 (BR3), which suggests that these receptors send signals that are nonredundant or that engage in crosstalk with each other. Here we show that BCR signaling induced production of the nonclassical transcription factor NF-kappaB pathway substrate p100, which is required for transmission of BR3 signals and thus B cell survival. The capacity for sustained p100 production emerged during transitional B cell differentiation, the stage at which BCR signals begin to mediate survival rather than negative selection. Our findings identify a molecular mechanism for the reliance of primary B cells on continuous BR3 and BCR signaling, as well as for the gradual resistance to negative selection that is acquired during B cell maturation.

Virulence of Toxoplasma gondii is associated with distinct dendritic cell responses and reduced numbers of activated CD8+ T cells.

The Toxoplasma gondii population consists of multiple strains, defined by genotype and virulence. Previous studies have established that protective immunity to this organism is mediated by IL-12, which drives T cells to produce IFN-gamma. Paradoxically, although type I and type II strains of T. gondii both induce IL-12 and IFN-gamma in the mouse, type I parasites are lethal, whereas type II strains establish chronic infection. The cellular basis for these strain-dependent differences remains unclear. To better understand these events, the CD8(+) T cell and dendritic cell (DC) responses to transgenic, OVA-expressing type I RH (RH OVA) and type II Prugniuad (Pru OVA) parasites were examined. Pru OVA-infected mice developed a robust DC response at the site of infection and the draining lymph node and generated a population of endogenous OVA-specific CD8(+) T cells. In contrast, RH OVA-infected mice had fewer DCs and OVA-specific CD8(+) T cells. RH OVA-infected mice given preactivated OVA-specific CD8(+) T cells were protected, suggesting that reduced DC-derived signals contributed to the low OVA-specific CD8(+) T cell numbers observed during type I infection. Indeed, DC depletion prior to Pru OVA infection resulted in a failure to generate activated OVA-specific CD8(+) T cells, and IL-12p70 treatment during RH OVA infection modestly increased the number of Ag-specific cells. Together, these data are consistent with a model of immunity to T. gondii in which strain-dependent DC responses shape the generation of Ag-specific CD8(+) T cells and determine the outcome of infection.

Role of the NF-κB transcription factor c-Rel in the generation of CD8+ T-cell responses to Toxoplasma gondii.

The nuclear factor κB transcription factor c-Rel is exclusively expressed in immune cells and plays a role in numerous cellular functions including proliferation, survival and production of chemokines and cytokines. c-Rel has also been implicated in the regulation of multiple genes involved in innate and adaptive immune responses to the intracellular protozoan parasite Toxoplasma gondii, in particular IL-12. To better understand how this transcription factor controls the CD8(+) T-cell response to this organism, wild-type (WT) and c-Rel(-/-) mice were challenged with a replication-deficient strain of T. gondii that expresses the model antigen ovalbumin (OVA). These studies revealed that c-Rel was required for optimal primary expansion of OVA-specific CD8(+) T cells and that immunized c-Rel-deficient mice were susceptible to challenge with a virulent strain of T. gondii. However, when c-Rel(-/-) cells specific for OVA were adoptively transferred into a WT recipient, or c-Rel(-/-) mice were treated with IL-12 at the time of immunization, there was no apparent proliferative defect. Surprisingly, upon secondary challenge, antigen-specific CD8(+) T cells in c-Rel(-/-) mice expanded to a much greater degree in terms of frequency as well as numbers when compared with WT mice. Despite this, the cytokine responses of c-Rel(-/-) mice remained defective, consistent with their susceptibility to secondary challenge. Together, these results indicate that in this infection model, the major influence of c-Rel in generation of CD8(+) T-cell responses is through its regulation of the inflammatory environment, rather than playing a substantial T-cell-intrinsic role.

Regulation of CD8+ T cell responses to infection with parasitic protozoa.

There are over 10,000 species of parasitic protozoa, a subset of which can cause considerable disease in humans. Here we examine in detail the complex immune response generated during infection with a subset of these parasites: Trypanosoma cruzi, Leishmania sp., Toxoplasma gondii, and Plasmodium sp. While these particular species perhaps represent the most studied parasites in terms of understanding how T cells function during infection, it is clear that the lessons learned from this body of work are also relevant to the other protozoa known to induce a CD8(+) T cell response. This review will highlight some of the key studies that established that CD8(+) T cells play a major role in protective immunity to protozoa, the factors that promote the generation as well as maintenance of the CD8(+) T cell response during these infections, and draw attention to some of the gaps in our knowledge. Moreover, the development of new tools, including MHC-Class I tetramer reagents and the use of TCR transgenic mice or genetically modified parasites, has provided a better appreciation of how parasite specific CD8(+) T cell responses are initiated and new insights into their phenotypic plasticity.

Kinetics and phenotype of vaccine-induced CD8+ T-cell responses to Toxoplasma gondii.

Multiple studies have established that the ability of CD8(+) T cells to act as cytolytic effectors and produce gamma interferon is important in mediating resistance to the intracellular parasite Toxoplasma gondii. To better understand the generation of the antigen-specific CD8(+) T-cell responses induced by T. gondii, mice were immunized with replication-deficient parasites that express the model antigen ovalbumin (OVA). Class I tetramers specific for SIINFEKL were used to track the OVA-specific endogenous CD8(+) T cells. The peak CD8(+) T-cell response was found at day 10 postimmunization, after which the frequency and numbers of antigen-specific cells declined. Unexpectedly, replication-deficient parasites were found to induce antigen-specific cells with faster kinetics than replicating parasites. The generation of optimal numbers of antigen-specific CD8(+) effector T cells was found to require CD4(+) T-cell help. At 7 days following immunization, antigen-specific cells were found to be CD62L(low), KLRG1(+), and CD127(low), and they maintained this phenotype for more than 70 days. Antigen-specific CD8(+) effector T cells in immunized mice exhibited potent perforin-dependent OVA-specific cytolytic activity in vivo. Perforin-dependent cytolysis appeared to be the major cytolytic mechanism; however, a perforin-independent pathway that was not mediated via Fas-FasL was also detected. This study provides further insight into vaccine-induced cytotoxic T-lymphocyte responses that correlate with protective immunity to T. gondii and identifies a critical role for CD4(+) T cells in the generation of protective CD8(+) T-cell responses.

Partial treatment interruption of protease inhibitors augments HIV-specific immune responses in vertically infected pediatric patients.

BACKGROUND: Although highly active antiretroviral therapy has significantly reduced morbidity and mortality in HIV-infected children, it often fails to completely suppress viral replication, thereby allowing the emergence of drug-resistant variants. Protease inhibitor (PI) based therapy has been hypothesized to depress cell-mediated immune responses by reducing antigen presentation. OBJECTIVES: To determine the effects of partial treatment interruption (PTI) of PI on HIV-specific cellular immune responses in children. METHODS: We conducted a retrospective longitudinal study of HIV-specific cellular immune responses in 13 children who were vertically infected with HIV. All had detectable plasma viremia and had undergone PTI for a median of 1.0 year (range, 0.41-3.35 years) while continuing nucleoside reverse transcriptase inhibitor and non-nucleoside reverse transcriptase inhibitor therapy. RESULTS: No significant changes in viral load were observed in the immediate time-point before and during PTI (P = 0.84) as well as in the overall period before and during PTI (P = 0.17). CD4 T-cell levels declined slowly immediately before and during PTI (P = 0.07) as well as during the overall PTI period (P = 0.0002), but the rate of CD4 T-cell decline was not significantly increased during PTI. Immediate to PTI, HIV-specific CD4 and CD8 T-cell responses increased by 70% (P < 0.0001) and 92% (P < 0.0001), respectively, and CD4 and CD8 T-cell activation levels (P = 0.6834 and P = 0.6081, respectively) remained unchanged. CONCLUSION: HIV-specific cellular immune responses are boosted in children who have interrupted PI-based therapy.

Subcellular antigen location influences T-cell activation during acute infection with Toxoplasma gondii.

Effective control of the intracellular protozoan parasite Toxoplasma gondii depends on the activation of antigen-specific CD8(+) T-cells that manage acute disease and prevent recrudescence during chronic infection. T-cell activation in turn, requires presentation of parasite antigens by MHC-I molecules on the surface of antigen presenting cells. CD8(+) T-cell epitopes have been defined for several T. gondii proteins, but it is unclear how these antigens enter into the presentation pathway. We have exploited the well-characterized model antigen ovalbumin (OVA) to investigate the ability of parasite proteins to enter the MHC-I presentation pathway, by engineering recombinant expression in various organelles. CD8(+) T-cell activation was assayed using 'B3Z' reporter cells in vitro, or adoptively-transferred OVA-specific 'OT-I' CD8(+) T-cells in vivo. As expected, OVA secreted into the parasitophorous vacuole strongly stimulated antigen-presenting cells. Lower levels of activation were observed using glycophosphatidyl inositol (GPI) anchored OVA associated with (or shed from) the parasite surface. Little CD8(+) T-cell activation was detected using parasites expressing intracellular OVA in the cytosol, mitochondrion, or inner membrane complex (IMC). These results indicate that effective presentation of parasite proteins to CD8(+) T-cells is a consequence of active protein secretion by T. gondii and escape from the parasitophorous vacuole, rather than degradation of phagocytosed parasites or parasite products.

CD8 T cell effector maturation in HIV-1-infected children.

HIV-1 infection generates maturational responses in overall CD4 and CD8 T cell populations in adults, with elevated expression of lytic effector molecules perforin and granzyme B, and reduced expression of CCR7 and CD45RA. Here, we have found that these marked effects were significantly less pronounced in children, both in terms of the skewed CCR7/CD45RA expression profile as well as the increased perforin expression. Similar to adults, HIV-specific CD8 cells in children were largely CD27+ CD45RA- and lacked perforin. However, one pediatric subject with late-stage infection displayed robust expansion of Gag 77-85-specific CD8 T cells which were perforin+ and lytic, but lacked expression of CD27 and IFNgamma. Our data indicate that the T cell effector maturation induced by HIV-1 infection is markedly weaker in children as compared to adults. The data also suggest, however, that the perforin-deficient state of HIV-specific CD8 T cells in children may be reversible.

HIV-1-specific CD4+ T cell responses in chronically HIV-1 infected blippers on antiretroviral therapy in relation to viral replication following treatment interruption.

The impact of transient viral load blips on anti-HIV-1 immune responses and on HIV-1 rebound following treatment interruption (TI) is not known. Clinical and immunological parameters were measured during 40 weeks of antiretroviral therapy (ART) and following TI in an observational cohort of 16 chronically HIV-1-infected subjects with or without observed viral load blips during ART. During therapy, blips in seven subjects were associated with higher anti-HIV-1 (p24) CD4+ T cell lymphoproliferative responses (p = 0.04), without a significant difference in T cell activation or total anti-HIV-1 CD8+ T cell interferon-gamma (IFN-gamma) responses when compared to nine matched non-blippers. Therapy interruption resulted in a significantly higher viral rebound in blippers by 8 week despite retention of higher lymphoproliferative p24 responses (p = 0.01) and a rise in CD3+ T cell activation (p = 0.04) and anti-HIV-1 CD8+ T cell responses in blippers by week 4 when compared to non-blippers. Past week 4 of interruption, therapy re-initiation criteria were also met by a higher frequency in blippers by week 14 (p < 0.04) with no difference between groups by week 24. These data support that blippers have higher anti-HIV lymphoproliferative responses while on ART but experience equal to higher viral rebound as compared to matched non-blippers upon TI.

Higher frequency of HIV-1-specific T cell immune responses in African American children vertically infected with HIV-1.

The progression of human immunodeficiency virus (HIV) disease and plasma levels of HIV may differ between racial groups. We compared HIV-specific T cell responses between vertically HIV-1-infected Hispanic and African American children. Subjects were matched for sex, age, viral load, and CD4(+) cell count in 18 pairs; T cell responses were measured by cytokine-enhanced interferon- gamma assay. Peripheral blood mononuclear cells were stimulated with HIV consensus peptides from Gag, Nef, and Tat. The influence of ethnicity, sex, age, viral load, and CD4(+) cell count on T cell responses was determined through linear regression analyses. After adjustment for CD4(+) count, age, and log(10) viral load, African American children demonstrated significantly higher Gag responses (average, 486 spot-forming cells higher; P=.01) than Hispanic children; this was significantly driven by robust responses in African American girls near the age of puberty, many of whom carried the human leukocyte antigen class I B*58 allele.

Development of innate CD4+ alpha-chain variable gene segment 24 (Valpha24) natural killer T cells in the early human fetal thymus is regulated by IL-7.

Natural killer (NK) T cells are innate CD1d-restricted immune cells involved in regulation of immune tolerance, tumor immunity, and immunity to infectious pathogens. Human alpha-chain variable gene segment 24 (Valpha24) NK T cells exist in the periphery as two functionally distinct subsets: one CD4+ and one CD4- subset. However, the developmental pathway of human Valpha24 NK T cells is not well understood. Here, we show that Valpha24 NK T cells develop in the fetal thymus. The relative number of intrathymic NK T cell precursors decline in a linear manner with gestational age, and they are very rare in the neonatal thymus, indicating that these cells preferentially develop in the early fetal thymus. Their restriction element, CD1d, is expressed by a vast majority of thymocytes. A majority of intrathymic Valpha24 NK T cell progenitors are CD4+, whereas a minority are CD4/8(+/+). CD4+ Valpha24 NK T cell precursors show features of mature NK T cells, such as high levels of their semiinvariant T cell receptor and CD3 and some expression of CD161, whereas the CD4/8(+/+) precursors seem less mature. The cytokine IL-7 shows a biphasic effect on Valpha24 NK T cell progenitors in fetal thymic organ culture, with high doses driving proliferation of immature CD161-progenitors and low doses supporting survival and maturation. Thus, the data demonstrate that human Valpha24 NK T cells of the CD4+, but not the CD4-, subset develop in the early fetal thymus. Furthermore, data suggest an intrathymic pathway of CD4+ Valpha24 NK T cell development that is regulated by IL-7.

Generation of CD3+CD8low thymocytes in the HIV type 1-infected thymus.

Infection with the HIV type 1 (HIV-1) can result both in depletion of CD4(+) T cells and in the generation of dysfunctional CD8(+) T cells. In HIV-1-infected children, repopulation of the peripheral T cell pool is mediated by the thymus, which is itself susceptible to HIV-1 infection. Previous work has shown that MHC class I (MHC I) molecules are strongly up-regulated as result of IFN-alpha secretion in the HIV-1-infected thymus. We demonstrate in this study that increased MHC I up-regulation on thymic epithelial cells and double-positive CD3(-/int)CD4(+)CD8(+) thymocytes correlates with the generation of mature single-positive CD4(-)CD8(+) thymocytes that have low expression of CD8. Treatment of HIV-1-infected thymus with highly active antiretroviral therapy normalizes MHC I expression and surface CD8 expression on such CD4(-)CD8(+) thymocytes. In pediatric patients with possible HIV-1 infection of the thymus, a low CD3 percentage in the peripheral circulation is also associated with a CD8(low) phenotype on circulating CD3(+)CD8(+) T cells. Furthermore, CD8(low) peripheral T cells from these HIV-1(+) pediatric patients are less responsive to stimulation by Ags from CMV. These data indicate that IFN-alpha-mediated MHC I up-regulation on thymic epithelial cells may lead to high avidity interactions with developing double-positive thymocytes and drive the selection of dysfunctional CD3(+)CD8(low) T cells. We suggest that this HIV-1-initiated selection process may contribute to the generation of dysfunctional CD8(+) T cells in HIV-1-infected patients.

HIV-specific CD8+ T cell function in children with vertically acquired HIV-1 infection is critically influenced by age and the state of the CD4+ T cell compartment.

The immunology of vertical HIV transmission differs from that of adult infection in that the immune system of the infant is not fully matured, and the factors that influence the functionality of CD8(+) T cell responses against HIV in children remain largely undefined. We have investigated CD8(+) T cell responses in 65 pediatric subjects with vertically acquired HIV-1 infection. Vigorous, broad, and Ag dose-driven CD8(+) T cell responses against HIV Ags were frequently observed in children who were older than 3 years of age and maintained CD4(+) T cell counts >400 cells/ micro l. In contrast, younger age or a CD4(+) T cell count <400 cells/ micro l was associated with poor CD8(+) T cell responses and high HIV loads. Furthermore, subjects with a severely depleted and phenotypically altered CD4(+) T cell compartment had circulating Gag-specific CD8(+) T cells with impaired IFN-gamma production. When viral load was not suppressed by antiviral treatment, subjects that fell below the putative age and CD4(+) T cell count thresholds had significantly reduced CD8(+) T cell responses and significantly higher viral loads. Thus, the data suggest that fully effective HIV-specific CD8(+) T cell responses take years to develop despite an abundance of Ag in early life, and responses are further severely impaired, independent of age, in children who have a depleted or skewed CD4(+) T cell compartment. The results are discussed in relation to differences between the neonatal and adult immune systems in the ability to respond to HIV infection.

Presence of human immunodeficiency virus-1-specific CD4 and CD8 cellular immune responses in children with full or partial virus suppression.

The present study assessed antiviral T cell immune responses in 48 human immunodeficiency virus (HIV)-infected children with a stable or decreasing CD4(+) T cell counts and different levels of viral control, in the presence or absence of antiretroviral therapy. Children with full (<40 copies/mL) or partial (<50,000 copies/mL) virus suppression and with a history of stable CD4(+) T cell counts had significantly increased levels of anti-HIV CD4(+) T cell lymphoproliferative responses, lower levels of CD38(+), and higher CD8(+)/CD28(+) T cell percentage, compared with those in treated children with a lack of virus suppression (>50,000 copies/mL). Levels of anti-HIV CD8(+) T cell activity, although higher in treated children with a lack of virus suppression, were not significantly different between the groups. Although levels of anti-HIV CD4(+) and CD8(+) T cell responses were not associated, these levels of responses were associated with the percentage of specific T cell subsets. Overall, a history of stable CD4(+) T cell counts, as a result of therapy that imparted full or partial virus suppression, was associated with increased levels of anti-HIV CD4(+) T helper responses and decreased T cell activation.