B cells are required for generation of protective effector and memory CD4 cells in response to Pneumocystis lung infection

B cell-deficient mice are susceptible to infection by Pneumocystis carinii f. sp. muris (PC). To determine whether this susceptibility is due to a requirement for B cells to prime T cells, we compared CD4 T cell responses to PC in bone marrow chimeric mice that express MHC class II (MHCII) on all APCs (wild-type (WT) chimeras) and in bone marrow chimeric mice that express MHCII on all APCs except B cells (MHCII(-/-) chimeras). Although PC was rapidly cleared by WT chimeric mice, PC levels remained high in chimeric mice that lacked MHCII on B cells. In addition, although T cells were primed in the draining lymph nodes of MHCII(-/-) chimeric mice, the number of activated CD4 T cells infiltrating the lungs of these mice was reduced relative to the number in the lungs of WT chimeras. We also adoptively transferred purified CD4 T cells from the draining lymph nodes of PC-infected normal or B cell-deficient mice into SCID mice. Mice that received CD4 cells from normal mice were able to mount a response to infection in the lungs and clear PC. However, mice that received CD4 cells from B cell-deficient mice had a delayed T cell response in the lungs and failed to control the infection. These data indicate that B cells play a vital role in generation of CD4(+) memory T cells in response to PC infection in the lungs.     

Type-I IFN signaling is required for the induction of antigen-specific CD8(+) T cell responses by adenovirus vector vaccine in the gut-mucosa

Microtubules in living cells are very important component for various cellular functions as well as to maintain the cell shape. Mechanical properties of microtubules play a vital role in their functions and structure. To understand the mechanical properties of microtubules in living cells, we developed an orthotropic-Pasternak model and investigated the vibrational behavior when microtubules are embedded in surrounding elastic medium. We considered microtubules as orthotropic elastic shell and its surrounding elastic matrix as Pasternak foundation. We found that due to mechanical coupling of microtubules with elastic medium, the flexural vibration is increased with the stiffening of elastic medium. We noticed that foundation modulus (H) and shear modulus (G) have more effect on radial vibrational mode as compared to longitudinal vibrational mode and torsional vibrational mode.     

CD4 is required for the development of a protective granulomatous response to pulmonary tuberculosis

To confirm the primary role of CD4 T cells in pulmonary tuberculosis, mice with a disruption of their CD4 gene (CD4 KO) were exposed to an aerosol of Mycobacterium tuberculosis and survival, cellular responses in the lung and granuloma development followed. CD8 and NK cells from the lungs of infected CD4 KO mice expressed IFN-gamma and were recruited in numbers similar to those seen in the C57BL/6 mice; recruitment correlated with initial control of bacteria. The major defect in mice lacking CD4 was the significant reduction in total cellular recruitment into the lungs. CD4 KO mice did not generate the typical mononuclear granulomatous lesions, instead the cellular influx was macrophage in character and was localized as perivascular cuffing. Early control of M. tuberculosis growth is therefore independent of CD4+ cells but such cells are required to ensure recruitment of mononuclear cells to the lung and thus ensure long-term survival.     

Type I IFN negatively regulates CD8+ T cell responses through IL-10-producing CD4+ T regulatory 1 cells

Pleiotropic, immunomodulatory effects of type I IFN on T cell responses are emerging. We used vaccine-induced, antiviral CD8(+) T cell responses in IFN-beta (IFN-beta(-/-))- or type I IFN receptor (IFNAR(-/-))-deficient mice to study immunomodulating effects of type I IFN that are not complicated by the interference of a concomitant virus infection. Compared with normal B6 mice, IFNAR(-/-) or IFN-beta(-/-) mice have normal numbers of CD4(+) and CD8(+) T cells, and CD25(+)FoxP3(+) T regulatory (T(R)) cells in liver and spleen. Twice as many CD8(+) T cells specific for different class I-restricted epitopes develop in IFNAR(-/-) or IFN-beta(-/-) mice than in normal animals after peptide- or DNA-based vaccination. IFN-gamma and TNF-alpha production and clonal expansion of specific CD8(+) T cells from normal and knockout mice are similar. CD25(+)FoxP3(+) T(R) cells down-modulate vaccine-primed CD8(+) T cell responses in normal, IFNAR(-/-), or IFN-beta(-/-) mice to a comparable extent. Low IFN-alpha or IFN-beta doses (500-10(3) U/mouse) down-modulate CD8(+) T cells priming in vivo. IFNAR- and IFN-beta-deficient mice generate 2- to 3-fold lower numbers of IL-10-producing CD4(+) T cells after polyclonal or specific stimulation in vitro or in vivo. CD8(+) T cell responses are thus subjected to negative control by both CD25(+)FoxP3(+) T(R) cells and CD4(+)IL-10(+) T(R1) cells, but only development of the latter T(R) cells depends on type I IFN.     

CD4 T cells are required for CD8 T cell survival during both primary and memory recall responses

The role of CD4 T cell help in primary and secondary CD8 T cell responses to infectious pathogens remains incompletely defined. The primary CD8 T response to infections was initially thought to be largely independent of CD4 T cells, but it is not clear why some primary, pathogen-specific CD8 T cell responses are CD4 T cell dependent. Furthermore, although the generation of functional memory CD8 T cells is CD4 T cell help dependent, it remains controversial when the "help" is needed. In this study, we demonstrated that CD4 T cell help was not needed for the activation and effector differentiation of CD8 T cells during the primary response to vaccinia virus infection. However, the activated CD8 T cells showed poor survival without CD4 T cell help, leading to a reduction in clonal expansion and a diminished, but stable CD8 memory pool. In addition, we observed that CD4 T cell help provided during both the primary and secondary responses was required for the survival of memory CD8 T cells during recall expansion. Our study indicates that CD4 T cells play a crucial role in multiple stages of CD8 T cell response to vaccinia virus infection and may help to design effective vaccine strategies.     

CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection

Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4(+) T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4(+) T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4(+) T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4(+) T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4(+) T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis.     

Type 1 IFN maintains the survival of anergic CD4+ T cells

Anergic T cells have immunoregulatory activity and can survive for extended periods in vivo. It is unclear how anergic T cells escape from deletion, because both anergy and apoptosis can occur after TCR ligation. Stimulation of human CD4+ T cell clones reactive to influenza hemagglutinin peptides can occur in the absence of APCs when MHC class II-expressing, activated T cells present peptide to each other. This T:T peptide presentation can induce CD95-mediated apoptosis, while the cells that do not die are anergic. We found that the death after peptide or anti-CD3 treatment of a panel of CD4+ T cell clones is blocked by IFN-beta secreted by fibroblasts and also by IFN-alpha. This increases cell recovery after stimulation, which is not due to T cell proliferation. This mechanism for apoptosis inhibition rapidly stops protein kinase C-delta translocation from the cytoplasm to the nucleus, which is an early event in the death process. A central observation was that CD4+ T cells that are rescued from apoptosis after T:T presentation of peptide by IFN-alphabeta remain profoundly anergic to rechallenge with Ag-pulsed APCs. However, anergized cells retain the ability to respond to IL-2, showing that they are nonresponsive but functional. The prevention of peptide-induced apoptosis in activated T cells by IFN-alphabeta is a novel mechanism that may enable the survival and maintenance of anergic T cell populations after TCR engagement. This has important implications for the persistence of anergic T cells with the potential for immunoregulatory function in vivo.     

CD28 is required for T cell activation and IFN-gamma production by CD4+ and CD8+ T cells in response to Trypanosoma cruzi infection

In the present study we evaluated the mechanisms behind the implication of the costimulatory molecule CD28 for the immune response against the intracellular protozoan parasite Trypanosma cruzi. Our results reveal a critical role for CD28 in the activation of both CD4+ and CD8+ T cells and induction of the effector mechanisms that ultimately mediate the control of parasite growth and pathogenesis in infected mice. CD28-deficient (CD28-/-) mice are highly susceptible to T. cruzi infection, presenting higher parasitemia and tissue parasitism, but less inflammatory cell infiltrate in the heart than C57Bl/6 wild-type (WT) mice. All the infected WT mice survived acute infection, whereas 100% of CD28-/- mice succumbed to it. The increased susceptibility of the CD28-/- mice was associated with a dramatic decrease in the production of IFN-gamma by both CD4+ and CD8+ T cells resulting in a diminished capacity to produce nitric oxide (NO) and mediate parasite killing. T cell activation was also profoundly impaired in CD28-/- mice, which presented decreased lymphoproliferative response after the infection compared to WT mice. Together, these data represent the first evidence that CD28 is critical for efficient CD4+ T cell activation in response to T. cruzi infection in mice.     

P38 MAP kinase signaling is required for the conversion of CD4+CD25- T cells into iTreg

CD4+CD25+ regulatory T cells (Treg) are important mediators of immune tolerance. A subset of Treg can be generated in the periphery by TGF-beta dependent conversion of conventional CD4+CD25- T cells into induced Treg (iTreg). In chronic viral infection or malignancy, such induced iTreg, which limit the depletion of aberrant or infected cells, may be of pathogenic relevance. To identify potential targets for therapeutic intervention, we investigated the TGF-beta signaling in Treg. In contrast to conventional CD4+ T cells, Treg exhibited marked activation of the p38 MAP kinase pathway. Inhibition of p38 MAP kinase activity prevented the TGF-beta-dependent conversion of CD4+CD25- T cells into Foxp3+ iTreg in vitro. Of note, the suppressive capacity of nTreg was not affected by inhibiting p38 MAP kinase. Our findings indicate that signaling via p38 MAP kinase seems to be important for the peripheral generation of iTreg; p38 MAP kinase could thus be a therapeutic target to enhance immunity to chronic viral infection or cancer.     

Cooperation between CD4 T helper cells is required for the generation of alloantigen-specific, IFN-gamma-producing human CD4 T cells

We have used MLR to assess the cell interactions that enable human PBL stimulated with irradiated, allogeneic PBL to give rise to allospecific, IFN-gamma-producing CD4 T cells. We were able to generate alloantigen-specific, IFN-gamma-producing T cells from peripheral blood. The responder and stimulator cell numbers required for the optimal generation of such cells, enumerated using an enzyme-linked immunospot assay, were separately determined for various combinations of responders and stimulators. The number of antigen-specific, IFN-gamma-producing cells generated per 10(6) responder cells, seeded at the initiation of culture, is critically dependent on the density of the responder cells, with minimal generation of IFN-gamma-producing T cells at low responder densities. These and further observations with fractionated responding PBL show that CD4 T-cell/CD4 T-cell interactions, which are completely independent of CD8() T cells, are necessary for the in vitro generation of alloantigen-specific, IFN-gamma-producing CD4 T cells.     

Monoubiquitinated histone H1B is required for antiviral protection in CD4(+)T cells resistant to HIV-1

Linker histone H1B (H1B) coeluted with an antiviral activity during the purification of HIV-1 resistance factor (HRF) from supernatants of HRF(+) cells. Western blot analysis of the supernatant using alpha-H1 and alpha-ubiquitin antibodies detected the same band of roughly 46 kDa; this band was absent from the control supernatant. Depletion of histone from biologically active material did not affect its potential, suggesting that ubiquitinated H1B is not required for the HRF-mediated antiviral protection in HIV-1 susceptible target cells; however, specific silencing of histone H1B via RNAi in HRF(+) cells reduced the biological activity of cell culture supernatants by 96% and reversed the HIV-1 resistance phenotype of HRF(+) cells. Exposure to HRF induced ubiquitination and secretion of H1B from target HIV-1 susceptible cells, suggesting that ubiquitinated H1B is a cofactor of HRF, possibly regulating its expression and secretion from CD4(+)T cells induced to resist HIV-1 infection.     

CD3-zeta surface expression is required for CD4-p56lck-mediated upregulation of T cell antigen receptor-CD3 signaling in T cells.

It has been proposed that during T cell receptor antigen recognition, CD4- or CD8-p56lck molecules interact with the T cell antigen receptor-CD3 complex (TCR-CD3) to phosphorylate various undefined substrates, which then initiate signal transduction through the TCR-CD3 complex. The ability of CD4 to modulate the TCR-CD3-induced increase in intracellular Ca2+, [Ca2+]i, and substrate tyrosine phosphorylation was studied in mutants of the human leukemic T cell line HPB-ALL characterized by their low expression of the TCR-CD3 complex on the cell surface. In TCR-CD3low cells, in which CD3-zeta was found to be associated with the TCR-CD3 complex, cross-linking CD3 with CD4 resulted in a profile of calcium mobilization, CD3-zeta, and phospholipase C-gamma 1 tyrosine phosphorylation similar to that observed in HPB-ALL cells, although the magnitude of generalized substrate tyrosine phosphorylation appeared to be smaller, as compared with wild-type cells. Responses were weak or absent when CD3 was cross-linked alone. In contrast, in a mutant in which association of CD3-zeta 2 with the TCR-CD3 was defective, cross-linking of CD3 with CD4 had a weaker effect on any of the activation parameters tested. These experiments showed that the presence of CD3-zeta 2 in the TCR-CD3 complex is of critical importance for the ability of CD4 to enhance early transducing signals inside the cell. The data also suggest that CD4-associated protein tyrosine kinase p56lck could up-regulate defective CD3-mediated induction of phospholipase C activity by increasing tyrosine phosphorylation of phospholipase C-gamma 1.     

Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells

Expression of the B‐cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B‐cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine‐based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B‐cell co‐receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igβ (CD79b), and the co‐receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igβ can be expressed on the B‐cell surface, where it is found in close proximity to CD19 and signals in an ITAM‐dependent manner. These data suggest that Igβ and CD19 are part of an alternative B‐cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells.     

Direct action of type I IFN on NK cells is required for their activation in response to vaccinia viral infection in vivo

Type I IFN plays an important role in the activation of NK cells. However, the mechanism underlying type I IFN-dependent NK cell activation remains largely unknown. A recent report suggested that type I IFN acted on accessory dendritic cells, leading to IL-15 production, and that subsequent trans-presentation of IL-15 was required for NK cell activation upon stimulation with synthetic TLR ligands. It is not clear how type I IFN regulates NK cell activation in response to live pathogens. Using a murine model of infection with vaccinia virus (VV), we previously demonstrated a critical role for type I IFN in the innate immune control of VV infection. In this study, we first showed that type I IFN did not directly protect L929 cells from VV infection in vitro and that type I IFN-dependent innate immune control of VV infection in vivo was mediated by activated NK cells. We further demonstrated that direct action of type I IFN on NK cells, but not on dendritic cells, is required for the activation of NK cells in response to VV infection both in vitro and in vivo, leading to efficient VV clearance. Our findings may help design effective strategies for the control of poxviral infections in vivo.     

CD18 is required for optimal development and function of CD4+CD25+ T regulatory cells

CD4+CD25+ T regulatory (Treg) cells inhibit immunopathology and autoimmune disease in vivo. CD4+CD25+ Treg cells' capacity to inhibit conventional T cells in vitro is dependent upon cell-cell contact; however, the cell surface molecules mediating this cell:cell contact have not yet been identified. LFA-1 (CD11a/CD18) is an adhesion molecule that plays an established role in T cell-mediated cell contact and in T cell activation. Although expressed at high levels on murine CD4+CD25+ Treg cells, the role of LFA-1 in these cells has not been defined previously. We hypothesized that LFA-1 may play a role in murine CD4+CD25+ Treg function. To evaluate this, we analyzed LFA-1-deficient (CD18-/-) CD4+CD25+ T cells. We show that CD18-/- mice demonstrate a propensity to autoimmunity. Absence of CD18 led to diminished CD4+CD25+ T cell numbers and affected both thymic and peripheral development of these cells. LFA-1-deficient CD4+CD25+ T cells were deficient in mediating suppression in vitro and in mediating protection from colitis induced by the transfer of CD4+CD25- T cells into lymphopenic hosts. Therefore, we define a crucial role for CD18 in optimal CD4+CD25+ Treg development and function.     

Cutting Edge: The direct action of type I IFN on CD4 T cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection

The action of type I IFN (IFN-I) on APCs is well studied, but their direct effect on CD4 T cells is unclear. To address this, we transferred IFN-I receptor-deficient (IFN-IR(0)) and -sufficient (wild-type, WT) TCR-transgenic CD4 T cells into WT mice and analyzed their response to immunization. In response to lymphocytic choriomeningitis virus immunization, WT CD4 T cells expanded approximately 100-fold, whereas IFN-IR(0) CD4 T cells expanded <10-fold. However, both WT and IFN-IR(0) CD4 T cells expanded approximately 10-fold after Listeria monocytogenes immunization. Poor expansion of IFN-IR(0) CD4 T cells after lymphocytic choriomeningitis virus immunization was not due to a defect in proliferation or initial activation but to poor survival of the daughter cells. Thus, direct IFN-I signals can play either a critical or minimal role in CD4 T cell clonal expansion depending on the specific pathogen.     

LFA-1 on CD4+ T cells is required for optimal antigen-dependent activation in vivo

The leukocyte-specific integrin, LFA-1, plays a critical role in trafficking of T cells to both lymphoid and nonlymphoid tissues. However, the role of LFA-1 in T cell activation in vivo has been less well understood. Although there have been reports describing LFA-1-deficient T cell response defects in vivo, due to impaired migration to lymphoid structures and to sites of effector function in the absence of LFA-1, it has been difficult to assess whether T cells also have a specific activation defect in vivo. We examined the role of LFA-1 in CD4(+) T cell activation in vivo by using a system that allows for segregation of the migration and activation defects through the adoptive transfer of LFA-1-deficient (CD18(-/-)) CD4(+) T cells from DO11.10 Ag-specific TCR transgenic mice into wild-type BALB/c mice. We find that in addition to its role in trafficking to peripheral lymph nodes, LFA-1 is required for optimal CD4(+) T cell priming in vivo upon s.c. immunization. CD18(-/-) DO11.10 CD4(+) T cells primed in the lymph nodes demonstrate defects in IL-2 and IFN-gamma production. In addition, recipient mice adoptively transferred with CD18(-/-) DO11.10 CD4(+) T cells demonstrate a defect in OVA-specific IgG2a production after s.c. immunization. The defect in priming of CD18(-/-) CD4(+) T cells persists even in the presence of proliferating CD18(+/-) CD4(+) T cells and in lymphoid structures to which there is no migration defect. Taken together, these results demonstrate that LFA-1 is required for optimal CD4(+) T cell priming in vivo.     

Type I IFN induced by adenovirus serotypes 28 and 35 has multiple effects on T cell immunogenicity

Recombinant adenovirus (rAd) vectors are being investigated as vaccine delivery vehicles in preclinical and clinical studies. rAds constructed from different serotypes differ in receptor usage, tropism, and ability to activate cells, aspects of which likely contribute to their different immunogenicity profiles. In this study, we compared the infectivity and cell stimulatory capacity of recombinant adenovirus serotype 5 (rAd5), recombinant adenovirus serotype 28 (rAd28), and recombinant adenovirus serotype 35 (rAd35) in association with their respective immunogenicity profiles. We found that rAd28 and rAd35 infected and led to the in vitro maturation and activation of both human and mouse dendritic cells more efficiently compared with rAd5. In stark contrast to rAd5, rAd28 and rAd35 induced production of IFN-α and stimulated IFN-related intracellular pathways. However, the in vivo immunogenicity of rAd28 and rAd35 was significantly lower than that of rAd5. Deletion of IFN-α signaling during vaccination with rAd28 and rAd35 vectors increased the magnitude of the insert-specific T cell response to levels induced by vaccination with rAd5 vector. The negative impact of IFN-α signaling on the magnitude of the T cell response could be overcome by increasing the vaccine dose, which was also associated with greater polyfunctionality and a more favorable long-term memory phenotype of the CD8 T cell response in the presence of IFN-α signaling. Taken together, our results demonstrate that rAd-induced IFN-α production has multiple effects on T cell immunogenicity, the understanding of which should be considered in the design of rAd vaccine vectors.     

Direct stimulation of T cells by type I IFN enhances the CD8+ T cell response during cross-priming

Type I IFN (IFN-alphabeta), which is produced rapidly in response to infection, plays a key role in innate immunity and also acts as a stimulus for the adaptive immune response. We have investigated how IFN-alphabeta induces cross-priming, comparing CD8+ T cell responses generated against soluble protein Ags in the presence or absence of IFN-alphabeta. Injection of IFN-alpha was found to prolong the proliferation and expansion of Ag-specific CD8+ T cells, which was associated with marked up-regulation of IL-2 and IL-15 receptors on Ag-specific cells and expression of IL-15 in the draining lymph node. Surprisingly, neither IL-2 nor IL-15 was required for IFN-alpha-induced cross-priming. Conversely, expression of the IFN-alphabetaR by T cells was shown to be necessary for effective stimulation of the response by IFN-alpha. The finding that T cells represent direct targets of IFN-alphabeta-mediated stimulation reveals an additional mechanism by which the innate response to infection promotes adaptive immunity.