Ablation of CD8 and CD4 T cell responses by high viral loads

To evaluate the impact of sustained viral loads on anti-viral T cell responses we compared responses that cleared acute lymphocytic choriomeningitis virus infection with those that were elicited but could not resolve chronic infection. During acute infection, as replicating virus was cleared, CD8 T cell responses were down-regulated, and a pool of resting memory cells developed. In chronically infected hosts, the failure to control the infection was associated with pronounced and prolonged activation of virus-specific CD8 T cells. Nevertheless, there was a progressive diminution of their effector activities as their capacity to produce first IL-2, then TNF-alpha, and finally IFN-gamma was lost. Chronic lymphocytic choriomeningitis virus infection was also associated with differential contraction of certain CD8 T cell responses, resulting in altered immunodominance. However, this altered immunodominance was not due to selective expansion of T cells expressing particular TCR Vbeta segments during chronic infection. High viral loads were not only associated with the ablation of CD8 T cell responses, but also with impaired production of IL-2 by virus-specific CD4 T cells. Taken together, our data show that sustained exposure to high viral loads results in the progressive functional inactivation of virus-specific T cell responses, which may further promote virus persistence.     

IRF4 Regulates the Ratio of T-Bet to Eomesodermin in CD8+ T Cells Responding to Persistent LCMV Infection

CD8⁺ T cell exhaustion commonly occurs in chronic infections and cancers. During T cell exhaustion there is a progressive and hierarchical loss of effector cytokine production, up-regulation of inhibitory co-stimulatory molecules, and eventual deletion of antigen specific cells by apoptosis. A key factor that regulates T cell exhaustion is persistent TCR stimulation. Loss of this interaction results in restoration of CD8⁺ T cell effector functions in previously exhausted CD8⁺ T cells. TCR stimulation is also important for the differentiation of Eomes^hi anti-viral CD8⁺ effector T cells from T-bet^hi precursors, both of which are required for optimal viral control. However, the molecular mechanisms regulating the differentiation of these two cell subsets and the relative ratios required for viral clearance have not been described. We show that TCR signal strength regulates the relative expression of T-bet and Eomes in antigen-specific CD8⁺ T cells by modulating levels of IRF4. Reduced IRF4 expression results in skewing of this ratio in the favor of Eomes, leading to lower proportions and numbers of T-bet⁺ Eomes^- precursors and poor control of LCMV-clone 13 infection. Manipulation of this ratio in the favor of T-bet restores the differentiation of T-bet⁺ Eomes^- precursors and the protective balance of T-bet to Eomes required for efficient viral control. These data highlight a critical role for IRF4 in regulating protective anti-viral CD8⁺ T cell responses by ensuring a balanced ratio of T-bet to Eomes, leading to the ultimate control of this chronic viral infection.     

Impaired T cell immunity in B cell-deficient mice following viral central nervous system infection

CD8(+) T cells are required to control acute viral replication in the CNS following infection with neurotropic coronavirus. By contrast, studies in B cell-deficient (muMT) mice revealed Abs as key effectors in suppressing virus recrudescence. The apparent loss of initial T cell-mediated immune control in the absence of B cells was investigated by comparing T cell populations in CNS mononuclear cells from infected muMT and wild-type mice. Following viral recrudescence in muMT mice, total CD8(+) T cell numbers were similar to those of wild-type mice that had cleared infectious virus; however, virus-specific T cells were reduced at least 3-fold by class I tetramer and IFN-gamma ELISPOT analysis. Although overall T cell recruitment into the CNS of muMT mice was not impaired, discrepancies in frequencies of virus-specific CD8(+) T cells were most severe during acute infection. Impaired ex vivo cytolytic activity of muMT CNS mononuclear cells, concomitant with reduced frequencies, implicated IFN-gamma as the primary anti viral factor early in infection. Reduced virus-specific CD8(+) T cell responses in the CNS coincided with poor peripheral expansion and diminished CD4(+) T cell help. Thus, in addition to the lack of Ab, limited CD8(+) and CD4(+) T cell responses in muMT mice contribute to the ultimate loss of control of CNS infection. Using a model of virus infection restricted to the CNS, the results provide novel evidence for a role of B cells in regulating T cell expansion and differentiation into effector cells.     

CD4+ T-Cell Help Is Required for Effective CD8+ T Cell-Mediated Resolution of Acute Viral Hepatitis in Mice

Cytotoxic CD8+ T cells are essential for the control of viral liver infections, such as those caused by HBV or HCV. It is not entirely clear whether CD4+ T-cell help is necessary for establishing anti-viral CD8+ T cell responses that successfully control liver infection. To address the role of CD4+ T cells in acute viral hepatitis, we infected mice with Lymphocytic Choriomeningitis Virus (LCMV) of the strain WE; LCMV-WE causes acute hepatitis in mice and is cleared from the liver by CD8+ T cells within about two weeks. The role of CD4+ T-cell help was studied in CD4+ T cell-lymphopenic mice, which were either induced by genetic deficiency of the major histocompatibility (MHC) class II transactivator (CIITA) in CIITA−/− mice, or by antibody-mediated CD4+ cell depletion. We found that CD4+ T cell-lymphopenic mice developed protracted viral liver infection, which seemed to be a consequence of reduced virus-specific CD8+ T-cell numbers in the liver. Moreover, the anti-viral effector functions of the liver-infiltrating CD8+ T cells in response to stimulation with LCMV peptide, notably the IFN-γ production and degranulation capacity were impaired in CIITA−/− mice. The impaired CD8+ T-cell function in CIITA−/− mice was not associated with increased expression of the exhaustion marker PD-1. Our findings indicate that CD4+ T-cell help is required to establish an effective antiviral CD8+ T-cell response in the liver during acute viral infection. Insufficient virus control and protracted viral hepatitis may be consequences of impaired initial CD4+ T-cell help.     

Mechanisms of central nervous system viral persistence: the critical role of antibody and B cells.

Contributions of humoral and cellular immunity in controlling neurotropic mouse hepatitis virus persistence within the CNS were determined in B cell-deficient J(H)D and syngeneic H-2(d) B cell+ Ab-deficient mice. Virus clearance followed similar kinetics in all mice, confirming initial control of virus replication by cellular immunity. Nevertheless, virus reemerged within the CNS of all Ab-deficient mice. In contrast to diminished T cell responses in H-2(b) B cell-deficient muMT mice, the absence of B cells or Ab in the H-2(d) mice did not compromise expansion, recruitment into the CNS, or function of virus-specific CD4+ and CD8+ T cells. The lack of B cells and lymphoid architecture thus appears to manifest itself on T cell responses in a genetically biased manner. Increasing viral load did not enhance frequencies or effector function of virus-specific T cells within the CNS, indicating down-regulation of T cell responses. Although an Ab-independent antiviral function of B cells was not evident during acute infection, the presence of B cells altered CNS cellular tropism during viral recrudescence. Reemerging virus localized almost exclusively to oligodendroglia in B cell+ Ab-deficient mice, whereas it also replicated in astrocytes in B cell-deficient mice. Altered tropism coincided with distinct regulation of CNS virus-specific CD4+ T cells. These data conclusively demonstrate that the Ab component of humoral immunity is critical in preventing virus reactivation within CNS glial cells. B cells themselves may also play a subtle role in modulating pathogenesis by influencing tropism.     

Role of PKR and Type I IFNs in Viral Control during Primary and Secondary Infection

Type I interferons (IFNs) are known to mediate viral control, and also promote survival and expansion of virus-specific CD8⁺ T cells. However, it is unclear whether signaling cascades involved in eliciting these diverse cellular effects are also distinct. One of the best-characterized anti-viral signaling mechanisms of Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. Here, we have investigated the role of PKR and Type I IFNs in regulating viral clearance and CD8⁺ T cell response during primary and secondary viral infections. Our studies demonstrate differential requirement for PKR, in viral control versus elicitation of CD8⁺ T cell responses during primary infection of mice with lymphocytic choriomeningitis virus (LCMV). PKR-deficient mice mounted potent CD8⁺ T cell responses, but failed to effectively control LCMV. The compromised LCMV control in the absence of PKR was multifactorial, and linked to less effective CD8⁺ T cell-mediated viral suppression, enhanced viral replication in cells, and lower steady state expression levels of IFN-responsive genes. Moreover, we show that despite normal expansion of memory CD8⁺ T cells and differentiation into effectors during a secondary response, effective clearance of LCMV but not vaccinia virus required PKR activity in infected cells. In the absence of Type I IFN signaling, secondary effector CD8⁺ T cells were ineffective in controlling both LCMV and vaccinia virus replication in vivo. These findings provide insight into cellular pathways of Type I IFN actions, and highlight the under-appreciated importance of innate immune mechanisms of viral control during secondary infections, despite the accelerated responses of memory CD8⁺ T cells. Additionally, the results presented here have furthered our understanding of the immune correlates of anti-viral protective immunity, which have implications in the rational design of vaccines.     

Murine cytomegalovirus interference with antigen presentation has little effect on the size or the effector memory phenotype of the CD8 T cell response

As with most herpesviruses, CMVs encode viral genes that inhibit Ag presentation to CD8 T cells (VIPRs). VIPR function has been assumed to be essential for CMV to establish its characteristic lifetime infection of its host. We compared infection of C57BL/6 mice with wild-type murine CMV (MCMV) and a virus lacking each of MCMV's three known VIPRs: m4, m6, and m152. During acute infection, there was very little difference between the two viruses with respect to the kinetics of viral replication and clearance, or in the size and kinetics of the virus-specific CD8 T cell response. During chronic infection, a large, effector memory, virus-specific CD8 T cell population (CD8(low)CD62L(-)CD11c(+)NKG2A(+)) was maintained in both infections; the size and phenotype of the CD8 T cell response to both viruses was remarkably similar. The characteristic effector memory phenotype of the CD8 T cells suggested that both wild-type and Deltam4+m6+m152 virus continued to present Ag to CD8 T cells during the chronic phase of infection. During the chronic phase of infection, MCMV cannot be isolated from immunocompetent mice. However, upon immunosuppression, both Deltam4+m6+m152 and wild-type virus could be reactivated from mice infected for 6 wk. Thus, restoring the ability of CD8 T cells to detect MCMV had little apparent effect on the course of MCMV infection and on the CD8 T cell response to it. These results challenge the notion that VIPR function is necessary for CMV persistence in the host.     

Contributions of CD8+ T cells and viral spread to demyelinating disease

Acute and chronic demyelination are hallmarks of CNS infection by the neurotropic JHM strain of mouse hepatitis virus. Although infectious virus is cleared by CD8+ T cells, both viral RNA and activated CD8+ T cells remain in the CNS during persistence potentially contributing to pathology. To dissociate immune from virus-mediated determinants initiating and maintaining demyelinating disease, mice were infected with two attenuated viral variants differing in a hypervariable region of the spike protein. Despite similar viral replication and tropism, one infection was marked by extensive demyelination and paralysis, whereas the other resulted in no clinical symptoms and minimal neuropathology. Mononuclear cells from either infected brain exhibited virus specific ex vivo cytolytic activity, which was rapidly lost during viral clearance. As revealed by class I tetramer technology the paralytic variant was superior in inducing specific CD8+ T cells during the acute disease. However, after infectious virus was cleared, twice as many virus-specific IFN-gamma-secreting CD8+ T cells were recovered from the brains of asymptomatic mice compared with mice undergoing demyelination, suggesting that IFN-gamma ameliorates rather than perpetuates JHM strain of mouse hepatitis virus-induced demyelination. The present data thus indicate that in immunocompetent mice, effector CD8+ T cells control infection without mediating either clinical disease or demyelination. In contrast, demyelination correlated with early and sustained infection of the spinal cord. Rapid viral spread, attributed to determinants within the spike protein and possibly perpetuated by suboptimal CD8+ T cell effector function, thus ultimately leads to the process of immune-mediated demyelination.     

Maintenance, loss, and resurgence of T cell responses during acute, protracted, and chronic viral infections

The acute phase of many viral infections is associated with the induction of a pronounced CD8 T cell response which plays a principle role in clearing the infection. By contrast, certain infections are not as readily controlled. In this study, we have used the well-defined system of lymphocytic choriomeningitis virus (LCMV) infection of mice to determine quantitative and qualitative changes in virus-specific CD8 T cell responses that rapidly resolve acute infections, more slowly control protracted infections, or fail to clear chronic infections. Acute LCMV infection elicits potent, functional, multi-epitope-specific CD8 T cell responses. Virus-specific CD8 T cells also expand, albeit to a lesser extent, during protracted LCMV infection. Under these conditions, there is a progressive diminution in the capacity to produce IL-2, TNF-alpha, and IFN-gamma. Changes in cytotoxic activities are also detectable but differ depending upon the specificity of the responding cells. As the infection is slowly resolved, a resurgence of cytokine production by virus-specific CD8 T cells is observed. CD4-deficient mice cannot control infection with certain strains of LCMV, but do mount multi-epitope-specific CD8 T cell responses that also lose effector capabilities; however, they are not maintained indefinitely in an unresponsive state as these cells become deleted over time. Overall, our findings suggest that constant high viral loads result in the progressive diminution of T cell effector functions and subsequent physical loss of the responding cells, whereas if the viral load is brought under control a partial restoration of CD8 T cell functions can occur.     

CD40-CD40 ligand costimulation is required for generating antiviral CD4 T cell responses but is dispensable for CD8 T cell responses.

This study documents a striking dichotomy between CD4 and CD8 T cells in terms of their requirements for CD40-CD40 ligand (CD40L) costimulation. CD40L-deficient (-/-) mice made potent virus-specific CD8 T cell responses to dominant as well as subdominant epitopes following infection with lymphocytic choriomeningitis virus. In contrast, in the very same mice, virus-specific CD4 T cell responses were severely compromised. There were 10-fold fewer virus-specific CD4 T cells in CD40L-/- mice compared with those in CD40L+/+ mice, and this inhibition was seen for both Th1 (IFN-gamma, IL-2) and Th2 (IL-4) responses. An in vivo functional consequence of this Th cell defect was the inability of CD40L-/- mice to control a chronic lymphocytic choriomeningitis virus infection. This study highlights the importance of CD40-CD40L interactions in generating virus-specific CD4 T cell responses and in resolving chronic viral infection.     

Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) clear infectious virus; nevertheless, virus persists in the CNS as noninfectious RNA, resulting in ongoing primary demyelination. Phenotypic and functional analysis of CNS infiltrating cells during acute infection revealed a potent regional CD8+ T cell response comprising up to 50% virus-specific T cells. The high prevalence of virus-specific T cells correlated with ex vivo cytolytic activity and efficient reduction in viral titers. Progressive viral clearance coincided with the loss of cytolytic activity, but retention of IFN-gamma secretion and increased expression of the early activation marker CD69, indicating differential regulation of effector function. Although the total number of infiltrating T cells declined following clearance of infectious virus, CD8+ T cells, both specific for the dominant viral epitopes and of unknown specificity, were retained within the CNS, suggesting an ongoing T cell response during persistent CNS infection involving a virus-independent component. Reversed immunodominance within the virus-specific CD8+ T cell population further indicated epitope-specific regulation, supporting ongoing T cell activation. Even in the absence of infectious virus, the CNS thus provides an environment that maintains both unspecific and Ag-specific CD8+ T cells with restricted effector function. Chronic T cell stimulation may thus play a role in preventing viral recrudescence, while increasing the risk of pathological conditions, such as demyelination.     

An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.     

CD8+ T cell responses in bronchoalveolar lavage fluid and peripheral blood mononuclear cells of infants with severe primary respiratory syncytial virus infections

A protective role for CD8+ T cells during viral infections is generally accepted, but little is known about how CD8+ T cell responses develop during primary infections in infants, their efficacy, and how memory is established after viral clearance. We studied CD8+ T cell responses in bronchoalveolar lavage (BAL) samples and blood of infants with a severe primary respiratory syncytial virus (RSV) infection. RSV-specific CD8+ T cells with an activated effector cell phenotype: CD27+CD28+CD45RO+CCR7-CD38+HLA-DR+Granzyme B+CD127- could be identified in BAL and blood. A high proportion of these CD8+ T cells proliferated and functionally responded upon in vitro stimulation with RSV Ag. Thus, despite the very young age of the patients, a robust systemic virus-specific CD8+ T cell response was elicited against a localized respiratory infection. RSV-specific T cell numbers as well as the total number of activated effector type CD8+ T cells peaked in blood around day 9-12 after the onset of primary symptoms, i.e., at the time of recovery. The lack of a correlation between RSV-specific T cell numbers and parameters of disease severity make a prominent role in immune pathology unlikely, in contrast the T cells might be involved in the recovery process.     

GITR Intrinsically Sustains Early Type 1 and Late Follicular Helper CD4 T Cell Accumulation to Control a Chronic Viral Infection

CD4 T cells are critical for control of persistent infections; however, the key signals that regulate CD4 T help during chronic infection remain incompletely defined. While several studies have addressed the role of inhibitory receptors and soluble factors such as PD-1 and IL-10, significantly less work has addressed the role of T cell co-stimulatory molecules during chronic viral infection. Here we show that during a persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13, mice lacking the glucocorticoid-induced tumor necrosis factor receptor related protein (GITR) exhibit defective CD8 T cell accumulation, increased T cell exhaustion and impaired viral control. Differences in CD8 T cells and viral control between GITR^+/+ and GITR^-/- mice were lost when CD4 T cells were depleted. Moreover, mixed bone marrow chimeric mice, as well as transfer of LCMV epitope-specific CD4 or CD8 T cells, demonstrated that these effects of GITR are largely CD4 T cell-intrinsic. GITR is dispensable for initial CD4 T cell proliferation and differentiation, but supports the post-priming accumulation of IFNγ⁺IL-2⁺ Th1 cells, facilitating CD8 T cell expansion and early viral control. GITR-dependent phosphorylation of the p65 subunit of NF-κB as well as phosphorylation of the downstream mTORC1 target, S6 ribosomal protein, were detected at day three post-infection (p.i.), and defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day five p.i. Consistently, we pinpoint IL-2-dependent CD4 T cell help for CD8 T cells to between days four and eight p.i. GITR also increases the ratio of T follicular helper to T follicular regulatory cells and thereby enhances LCMV-specific IgG production. Together, these findings identify a CD4 T cell-intrinsic role for GITR in sustaining early CD8 and late humoral responses to collectively promote control of chronic LCMV clone 13 infection.     

Immunoproteasome-deficient mice mount largely normal CD8+ T cell responses to lymphocytic choriomeningitis virus infection and DNA vaccination

During viral infection, constitutive proteasomes are largely replaced by immunoproteasomes, which display distinct cleavage specificities, resulting in different populations of potential CD8(+) T cell epitope peptides. Immunoproteasomes are believed to be important for the generation of many viral CD8(+) T cell epitopes and have been implicated in shaping the immunodominance hierarchies of CD8(+) T cell responses to influenza virus infection. However, it remains unclear whether these conclusions are generally applicable. In this study we investigated the CD8(+) T cell responses to lymphocytic choriomeningitis virus infection and DNA immunization in wild-type mice and in mice lacking the immunoproteasome subunits LMP2 or LMP7. Although the total number of virus-specific cells was lower in LMP2 knockout mice, consistent with their having lower numbers of naive cells before infection, the kinetics of virus clearance were similar in all three mouse strains, and LMP-deficient mice mounted strong primary and secondary lymphocytic choriomeningitis virus-specific CD8(+) T cell responses. Furthermore, the immunodominance hierarchy of the four investigated epitopes (nuclear protein 396 (NP(396)) > gp33 > gp276 > NP(205)) was well maintained. We observed a slight reduction in the NP(205)-specific response in LMP2-deficient mice, but this had no demonstrable biological consequence. DNA vaccination of LMP2- and LMP7-deficient mice induced CD8(+) T cell responses that were slightly lower than, although not significantly different from, those induced in wild-type mice. Taken together, our results challenge the notion that immunoproteasomes are generally needed for effective antiviral CD8(+) T cell responses and for the shaping of immunodominance hierarchies. We conclude that the immunoproteasome may affect T cell responses to only a limited number of viral epitopes, and we propose that its main biological function may lie elsewhere.     

Characterization of the Treg Response in the Hepatitis B Virus Hydrodynamic Injection Mouse Model

Regulatory T cells (Tregs) play an important role in counter-regulating effector T cell responses in many infectious diseases. However, they can also contribute to the development of T cell dysfunction and pathogen persistence in chronic infections. Tregs have been reported to suppress virus-specific T cell responses in hepatitis B virus (HBV) infection of human patients as well as in HBV animal models. However, the phenotype and expansion of Tregs has so far only been investigated in other infections, but not in HBV. We therefore performed hydrodynamic injections of HBV plasmids into mice and analyzed the Treg response in the spleen and liver. Absolute Treg numbers significantly increased in the liver but not the spleen after HBV injection. The cells were natural Tregs that surprisingly did not show any activation or proliferation in response to the infection. However, they were able to suppress effector T cell responses, as selective depletion of Tregs significantly increased HBV-specific CD8+ T cell responses and accelerated viral antigen clearance. The data implies that natural Tregs infiltrate the liver in HBV infection without further activation or expansion but are still able to interfere with T cell mediated viral clearance.     

Effect of Staphylococcus enterotoxin B on the concurrent CD8(+) T cell response to influenza virus infection

Bacterial superantigens have potent in vivo effects. Respiratory viral infections are often associated with secondary bacterial infections, raising the likelihood of exposure to bacterial superantigens after the initiation of the anti-viral immune response. In this study, the general and V beta-specific effects of exposure to Staphylococcal enterotoxin B (SEB) during influenza virus infection on both the ongoing acute and the subsequent recall CD8(+) T cell responses were analyzed, using the well-characterized murine influenza model system and tetrameric MHC/peptide reagents to directly identify virus-specific T cells. The results show that although superantigen exposure during the primary viral infection caused delayed viral clearance, there was remarkably little effect of SEB on the magnitude or TCR repertoire of the ongoing cytolytic T cell response or on the recall response elicited by secondary viral infection. Thus, despite the well-characterized immunomodulatory effects of SEB, there was surprisingly little interference with concurrent anti-viral immunity.     

Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection

The complement cascade defines an important link between the innate and the specific immune system. Here we show that mice deficient for the third component of complement (C3-/- mice) are highly susceptible to primary infection with influenza virus. C3-/- mice showed delayed viral clearance and increased viral titers in lung, whereas mice deficient for complement receptors CR1 and CR2 (Cr2-/- mice) cleared the infection normally. Priming of T-helper cells and cytotoxic T cells (CTLs) in lung-draining lymph nodes was reduced, and the recruitment into the lung of virus-specific CD4+ and CD8+ effector T cells producing interferon-gamma was severely impaired in C3-/- but not in Cr2-/- mice. Consequently, T-helper cell-dependent IgG responses were reduced in C3-/- mice but remained intact in Cr2-/- mice. These results demonstrate that complement induces specific immunity by promoting T-cell responses.