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.     

B7 costimulation is critical for antibody class switching and CD8(+) cytotoxic T-lymphocyte generation in the host response to vesicular stomatitis virus

Antibody and cytotoxic T-lymphocyte (CTL) responses have critical roles in eliminating many viral infections. In addition to stimulation of the T-cell receptor, T cells require costimulatory signals to respond optimally. We evaluated the role of B7 costimulatory molecules (B7-1 and B7-2) in the immune response to viral infection using vesicular stomatitis virus (VSV) and mice lacking either B7-1 or B7-2 or both molecules. Mice lacking both B7-1 and B7-2 had essentially no anti-VSV immunoglobulin G1 (IgG1) response, decreased IgG2a responses, and normal IgM responses, while mice lacking either B7-1 or B7-2 had unaltered anti-VSV antibody responses compared to wild-type mice. Depletion of CD4(+) cells further reduced the IgG2a response in mice lacking both B7 molecules, suggesting that CD4(-) cells may supply help for IgG2a in the absence of B7 costimulation. The absence of both B7 molecules profoundly reduced generation of both primary and secondary VSV-specific class I major histocompatibility complex (MHC)-restricted CTL, whereas VSV-specific CTL responses in mice lacking either B7-1 or B7-2 were similar to those of wild-type animals. Class I MHC-restricted CTL in wild-type mice were not dependent on CD4(+) cells, suggesting that the failure of CTL in the absence of B7s is due to a lack of B7 costimulation directly to the CD8(+) CTL. These data demonstrate that B7-1 and B7-2 have critical, overlapping functions in the antibody and CTL responses to this viral infection.     

4-1BB ligand, a member of the TNF family, is important for the generation of antiviral CD8 T cell responses.

4-1BB (CD137) is a costimulatory molecule expressed on activated T cells and interacts with 4-1BB ligand (4-1BBL) on APCs. To investigate the role of 4-1BB costimulation for the development of primary immune responses, 4-1BBL-deficient (4-1BBL-/-) mice were infected with lymphocytic choriomeningitis virus (LCMV). 4-1BBL-/- mice were able to generate CTL and eliminate acute LCMV infection with normal kinetics, but CD8 T cell expansion was 2- to 3-fold lower than in wild-type (+/+) mice. In the same mice, virus-specific CD4 Th and B cell responses were minimally affected, indicating that 4-1BB costimulation preferentially affects CD8 T cell responses. This result contrasts with our earlier work with CD40L-deficient (CD40L-/-) mice, in which the CD8 T cell response was unaffected and the CD4 T cell response was markedly impaired. When both 4-1BBL- and B7-dependent signals were absent, CD8 T cell expansion was further reduced, resulting in lower CTL activity and impairing their ability to clear LCMV. Altogether, these results indicate that T cells have distinct costimulatory requirements: optimal CD8 responses require 4-1BBL-dependent interactions, whereas CD4 responses are minimally affected by 4-1BB costimulation, but require CD40-CD40L and B7-dependent interactions.     

The role of CD40-CD154 interaction in antiviral T cell-independent IgG responses

Polyomavirus (PyV) infection elicits protective T cell-independent (TI) IgG responses in T cell-deficient mice. The question addressed in this report is whether CD40 signaling plays a role in this TI antiviral IgG response. Because CD40 ligand (CD40L) can be expressed on numerous cell types in addition to activated T cells, it is possible that cells other than T cells provide CD40L to signal through CD40 on B cells and hence positively influence the antiviral TI IgG responses. In this study we show, by blocking CD40-CD40L interactions in vivo with anti-CD40L Ab treatment in TCR betaxdelta-/- mice and by using SCID mice reconstituted with CD40-/- B cells, that the lack of CD40 signaling in B cells results in a 50% decrease in TI IgG secreted in response to PyV. SCID mice reconstituted with CD40L-/- B cells also responded to PyV infection with diminished IgG secretion compared with that of SCID mice reconstituted with wild-type B cells. This finding suggests that B cells may provide the CD40L for CD40 signaling in the absence of T cell help during acute virus infection. Our studies demonstrate that, although about half of the TI IgG responses to PyV are independent of CD40-CD40L interactions, these interactions occur in T cell-deficient mice and enhance antiviral TI Ab responses.     

Mechanism of reduced T-cell effector functions and class-switched antibody responses to herpes simplex virus type 2 in the absence of B7 costimulation

T-cell costimulation molecules B7-1 and B7-2 play an important role in activation of T cells to cytolytic effector function and production of cytokines. Interaction with B7 also causes T cells to upregulate surface molecules, such as CD40L, that effectively stimulate antibody responses in conjunction with cytokines. We have shown that mice lacking both B7-1 and B7-2 (B7KO mice), when infected intravaginally with virulent herpes simplex virus type 2 (HSV-2), developed more severe disease and higher mortality than their wild-type counterparts. We have now investigated the effects of B7 costimulation deficiency on induction of immune responses to HSV-2 infection of the genital tract. Fewer gamma interferon (IFN-gamma)-producing T cells were present in the genital lymph nodes of B7KO mice compared to wild-type mice, either acutely after primary infection or in recall responses. Less IFN-gamma and especially interleukin-10 were produced by B7KO mice, and cytolytic T-lymphocyte activity was also attenuated. Reduced expression of CD25 on CD4(+) T cells after infection of B7KO mice was consistent with deficits in T-cell activation to effector functions. Although HSV-specific immunoglobulin M (IgM) titers were comparable for both B7KO mice and wild-type mice, B7KO mice had significant deficits in HSV-specific serum IgG responses, with markedly reduced levels of IgG2a and IgG1. In addition, significantly less IgG was detected in the vaginal secretions of B7KO mice than in those from wild-type mice. CD4(+) T-cell expression of CD40L was depressed in B7KO mice in vivo and in vitro. Together with reduced cytokine production, these results suggest a mechanism for decreased IgG class switching or production. Thus, in the absence of B7 costimulation, na?ve T cells fail to undergo proper activation in response to HSV-2, which limits T-cell cytokine production, cytotoxic T lymphocyte activity, and provision of help for class-switched antibody responses.     

Role of CD40 ligand and CD28 in induction and maintenance of antiviral CD8+ effector T cell responses

The primary aim of this report was to evaluate the immune responses of CD40 ligand-deficient (CD40L-/-) mice infected with two viruses known to differ markedly in their capacity to replicate in the host. Lymphocytic choriomeningitis virus (LCMV) is a natural mouse pathogen that replicates widely and extensively, whereas vesicular stomatitis virus (VSV) spreads poorly. We found that the primary response of CD40L-/- mice toward VSV is significantly impaired; proliferation of both CD4+ and CD8+ cells is reduced 2- to 3-fold, few CD8+ cells acquire an activated phenotype, and little functional activity is induced. Very similar results were obtained in VSV-infected, CD28-deficient mice. In contrast, neither CD40L nor CD28 was required for induction of a primary CD8+ response toward LCMV. Surprisingly, lack of CD4+ T cells had no impact on the primary immune response toward any of the viruses, even though the CD40 ligand dependence demonstrated for VSV would be expected to be associated with CD4 dependence. Upon coinfection of VSV-infected mice with LCMV, the requirement for CD40 ligand (but not CD28) could be partially bypassed, as evidenced by a 3-fold increase in the frequency of VSV-specific CD8+ T cells on day 6 postinfection. Finally, despite the fact that the primary LCMV-specific CD8+ response is virtually unimpaired in CD40L-/- mice, their capacity to maintain CD8+ effector activity and to permanently control the infection is significantly reduced. Thus, our results demonstrate that the importance of CD40/CD40L interaction for activation of CD8+ T cells varies between viruses and over time.     

Antiviral immune responses in mice deficient for both interleukin-2 and interleukin-4.

Antiviral immune responses of mice lacking interleukin-2 (IL-2) or IL-4 or both IL-2 and IL-4 (IL-2/4) were compared by using different viruses. Primary cytotoxic T-lymphocyte (CTL) responses against lymphocytic choriomeningitis virus (LCMV) were only moderately reduced in mice lacking IL-2 and were normal in mice lacking IL-4. Mice deficient in both interleukins exhibited variable and more strongly reduced but nevertheless in vivo protective LCMV-specific CTL responses. Similar results were obtained with vaccinia virus. Upon virus-specific restimulation in vitro, spleen cells from IL-2- and IL-2/4-deficient mice failed to generate CTL responses against virus-infected target cells, whereas the response of mice deficient in only IL-4 was comparable to that of control mice. The addition of IL-2 during in vitro restimulation completely restored the responses of both IL-2 and IL-2/4-deficient mice. T-helper-cell-independent immunoglobulin M and T-helper-cell-dependent immunoglobulin G antibody responses against vesicular stomatitis virus glycoprotein were within normal ranges for the various mutant mice. After LCMV infection, specific antibody responses against LCMV nucleoprotein were reduced four- to eightfold. These results show that mice lacking IL-2/4 have an overall tendency to exhibit more severely reduced CTL responses than IL-2- or IL-4-deficient mice. Nevertheless, and surprisingly, in vivo protective immune responses were mounted in the absence of IL-2/4, suggesting that besides a minor contribution from IL-4, other interleukins compensate in vivo for the lack of IL-2 in IL-2-deficient mice.     

Antiviral CD4 and CD8 T-cell memory: differences in the size of the response and activation requirements

Following acute lymphocytic choriomeningitis virus (LCMV) infection, there is a potent antiviral CD8 T-cell response that eliminates the infection. This initial CD8 T-cell response is followed by a period of memory during which elevated numbers of virus-specific CD8 T cells remain in the mouse. CD4 T cells are also activated after LCMV infection, but relatively less is known about the magnitude and duration of the CD4 response. In this study, we used intracellular staining for interferon-gamma to measure both CD4 and CD8 responses in the same mice at the single cell level. After LCMV infection, there was an increase in the number of activated CD4 T cells and an associated increase in the number of virus-specific CD4 T cells. At the peak of this expansion phase, the frequency of virus-specific CD4 T cells was 1 in 20 (0.5-1.0 x 10(6) per spleen). Like the CD8 response, long-term CD4 memory could be found up to a year after the infection with frequencies of approximately 1 in 260 (0.5-1.5 x 10(5) per spleen). However, the magnitude of virus-specific CD8 T cells was greater than virus-specific CD4 T cells during all phases of the immune response (expansion, death, and memory). At day 8, there were 20- to 35-fold more virus-specific CD8 T cells than CD4 T cells. This initial difference in cell number lasted into the memory phase as there remained a ten- to 20-fold difference in the CD8 and CD4 responses. These results highlight the importance of the expansion phase in determining the size of the memory T-cell pool. In addition to the difference in the magnitude, the activation requirements of CD8 and CD4 T-cell responses were different: CD8 T responses were not affected by blockade of CD40-CD40 ligand interaction whereas CD4 responses were reduced 90%. So while there is long-term memory in both the CD8 and CD4 compartments, the rules regulating the activation of CD8 and CD4 T cells and the overall magnitude of the responses are different.     

CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection.

In this study, we have examined the relative contributions of CD4+ and CD8+ T cells in controlling an acute or chronic lymphocytic choriomeningitis virus (LCMV) infection. To study acute infection, we used the LCMV Armstrong strain, which is cleared by adult mice in 8 to 10 days, and to analyze chronic infection, we used a panel of lymphocyte-tropic and macrophage-tropic variants of LCMV that persist in adult mice for several months. We show that CD4+ T cells are not necessary for resolving an acute LCMV infection. CD4+ T-cell-depleted mice were capable of generating an LCMV-specific CD8+ cytotoxic T-lymphocyte (CTL) response and eliminated virus with kinetics similar to those for control mice. The CD8+ CTL response was critical for resolving this infection, since beta 2-microglobulin knockout (CD8-deficient) mice were unable to control the LCMV Armstrong infection and became persistently infected. In striking contrast to the acute infection, even a transient depletion of CD4+ T cells profoundly affected the outcome of infection with the macrophage- and lymphocyte-tropic LCMV variants. Adult mice given a single injection of anti-CD4 monoclonal antibody (GK1.5) at the time of virus challenge became lifelong carriers with high levels of virus in most tissues. Unmanipulated adult mice infected with the different LCMV variants contained virus for prolonged periods (> 3 months) but eventually eliminated infection from most tissues, and all of these mice had LCMV-specific CD8+ CTL responses. Although the level of CTL activity was quite low, it was consistently present in all of the chronically infected mice that eventually resolved the infection. These results clearly show that even in the presence of an overwhelming viral infection of the immune system, CD8+ CTL can remain active for long periods and eventually resolve and/or keep the virus infection in check. In contrast, LCMV-specific CTL responses were completely lost in chronically infected CD4-depleted mice. Taken together, these results show that CD4+ T cells are dispensable for short-term acute infection in which CD8+ CTL activity does not need to be sustained for more than 2 weeks. However, under conditions of chronic infection, in which CD8+ CTLs take several months or longer to clear the infection, CD4+ T-cell function is critical. Thus, CD4+ T cells play an important role in sustaining virus-specific CD8+ CTL during chronic LCMV infection. These findings have implications for chronic viral infections in general and may provide a possible explanation for the loss of human immunodeficiency virus-specific CD8+ CTL activity that is seen during the late stages of AIDS, when CD4+ T cells become limiting.     

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.     

The cytotoxic T-lymphocyte response to Sendai virus is unimpaired in the absence of gamma interferon.

Sendai virus is eliminated from the respiratory tract of gamma interferon (IFN-gamma) -/- BALB/c mice with normal kinetics. The level of virus-specific cytotoxic T-lymphocyte (CTL) activity in the cell population recovered by bronchoalveolar lavage is unimpaired, the prevalence of interleukin-4 (IL-4)-producing cells is increased, and the titers of virus-specific immunoglobulins IgG1 and IgG2b are higher in the IFN-gamma -/- mice. The emergence of this T-helper 2 response profile in both lymphoid tissue and the pneumonic lung has no obvious deleterious consequences. Virus clearance is slightly delayed following depletion of the CD4+ subset, with the effect being similar in magnitude for IFN-gamma -/- and +/+ mice. However, the generation of CTL precursors (CTLp) is diminished in the IFN-gamma -/- (but not +/+) mice in the absence of concurrent CD4+ T help. Apparently the clonal expansion of the CTLp population can be promoted either by a cytokine (perhaps IL-2) produced by the IFN-gamma -/- CD4+ T cells or by IFN-gamma made by other cell types in the +/+ mice.     

CD4 T cell-independent antibody response promotes resolution of primary influenza infection and helps to prevent reinfection

It is generally believed that the production of influenza-specific IgG in response to viral infection is dependent on CD4 T cells. However, we previously observed that CD40-deficient mice generate influenza-specific IgG during a primary infection, suggesting that influenza infection may elicit IgG responses independently of CD4 T cell help. In the present study, we tested this hypothesis and show that mice lacking CD40 or CD4 T cells produce detectable titers of influenza-specific IgG and recover from influenza infection in a manner similar to that of normal mice. In contrast, mice completely lacking B cells succumb to influenza infection, despite the presence of large numbers of functional influenza-specific CD8 effector cells in the lungs. Consistent with the characteristics of a T-independent Ab response, long-lived influenza-specific plasma cells are not found in the bone marrow of CD40-/- and class II-/- mice, and influenza-specific IgG titers wane within 60 days postinfection. However, despite the short-lived IgG response, CD40-/- and class II-/- mice are completely protected from challenge infection with the same virus administered within 30 days. This protection is mediated primarily by B cells and Ab, as influenza-immune CD40-/- and class II-/- mice were still resistant to challenge infection when T cells were depleted. These data demonstrate that T cell-independent influenza-specific Ab promotes the resolution of primary influenza infection and helps to prevent reinfection.     

Extralymphatic virus sanctuaries as a consequence of potent T-cell activation

T helper cells can support the functions of CD8(+) T cells against persistently infecting viruses such as murine lymphocytic choriomeningitis virus (LCMV), cytomegalovirus, hepatitis C virus and HIV. These viruses often resist complete elimination and remain detectable at sanctuary sites, such as the kidneys and other extralymphatic organs. The mechanisms underlying this persistence are not well understood. Here we show that mice with potent virus-specific T-cell responses have reduced levels and delayed formation of neutralizing antibodies, and these mice fail to clear LCMV from extralymphatic epithelia. Transfer of virus-specific B cells but not virus-specific T cells augmented virus clearance from persistent sites. Virus elimination from the kidneys was associated with the formation of IgG deposits in the interstitial space, presumably from kidney-infiltrating B cells. CD8(+) T cells in the kidneys of mice that did not clear virus from this site were activated but showed evidence of exhaustion. Thus, we conclude that in this model of infection, site-specific virus persistence develops as a consequence of potent immune activation coupled with reductions in virus-specific neutralizing antibodies. Our results suggest that sanctuary-site formation depends both on organ anatomy and on the induction of different adaptive immune effector mechanisms. Boosting T-cell responses alone may not reduce virus persistence.     

Host differences in influenza-specific CD4 T cell and B cell responses are modulated by viral strain and route of immunization

The antibody response to influenza infection is largely dependent on CD4 T cell help for B cells. Cognate signals and secreted factors provided by CD4 T cells drive B cell activation and regulate antibody isotype switching for optimal antiviral activity. Recently, we analyzed HLA-DR1 transgenic (DR1) mice and C57BL/10 (B10) mice after infection with influenza virus A/New Caledonia/20/99 (NC) and defined epitopes recognized by virus-specific CD4 T cells. Using this information in the current study, we demonstrate that the pattern of secretion of IL-2, IFN-γ, and IL-4 by CD4 T cells activated by NC infection is largely independent of epitope specificity and the magnitude of the epitope-specific response. Interestingly, however, the characteristics of the virus-specific CD4 T cell and the B cell response to NC infection differed in DR1 and B10 mice. The response in B10 mice featured predominantly IFN-γ-secreting CD4 T cells and strong IgG2b/IgG2c production. In contrast, in DR1 mice most CD4 T cells secreted IL-2 and IgG production was IgG1-biased. Infection of DR1 mice with influenza PR8 generated a response that was comparable to that in B10 mice, with predominantly IFN-γ-secreting CD4 T cells and greater numbers of IgG2c than IgG1 antibody-secreting cells. The response to intramuscular vaccination with inactivated NC was similar in DR1 and B10 mice; the majority of CD4 T cells secreted IL-2 and most IgG antibody-secreting cells produced IgG2b or IgG2c. Our findings identify inherent host influences on characteristics of the virus-specific CD4 T cell and B cell responses that are restricted to the lung environment. Furthermore, we show that these host influences are substantially modulated by the type of infecting virus via the early induction of innate factors. Our findings emphasize the importance of immunization strategy for demonstrating inherent host differences in CD4 T cell and B cell responses.     

CD40-CD40 ligand interactions promote trafficking of CD8+ T cells into the brain and protection against West Nile virus encephalitis

Recent studies have established a protective role for T cells during primary West Nile virus (WNV) infection. Binding of CD40 by CD40 ligand (CD40L) on activated CD4+ T cells provides an important costimulatory signal for immunoglobulin class switching, antibody affinity maturation, and priming of CD8+ T-cell responses. We examined here the function of CD40-dependent interactions in limiting primary WNV infection. Compared to congenic wild-type mice, CD40(-/-) mice uniformly succumbed to WNV infection. Although CD40(-/-) mice produced low levels of WNV-specific immunoglobulin M (IgM) and IgG, viral clearance from the spleen and serum was not altered, and CD8+ T-cell priming in peripheral lymphoid tissues was normal. Unexpectedly, CD8+ T-cell trafficking to the central nervous system (CNS) was markedly impaired in CD40(-/-) mice, and this correlated with elevated WNV titers in the CNS and death. In the brains of CD40(-/-) mice, T cells were retained in the perivascular space and did not migrate into the parenchyma, the predominant site of WNV infection. In contrast, in wild-type mice, T cells trafficked to the site of infection in neurons. Beside its role in maturation of antibody responses, our experiments suggest a novel function of CD40-CD40L interactions: to facilitate T-cell migration across the blood-brain barrier to control WNV infection.     

Suppression of virus-specific antibody production by CD8+ class I-restricted antiviral cytotoxic T cells in vivo.

The question of whether virus-induced immunosuppression includes the antibody response against the infecting virus itself was evaluated in a model situation. Transgenic mice expressing the T-cell receptor (TCR) specific for peptide 32-42 of lymphocytic choriomeningitis virus (LCMV) glycoprotein 1 presented by Db reacted with a strong transgenic cytotoxic T-lymphocyte (CTL) response starting on day 3 after infection with a high dose (10(6) PFU intravenously [i.v.]) of the WE strain of LCMV (LCMV-WE); LCMV-specific antibody production in the spleen was suppressed in these mice. Low-dose (10(2) PFU i.v.) infection resulted in an antiviral antibody response comparable to that of the transgene-negative littermates. The induction of suppression of LCMV-specific antibody responses was specifically mediated by CD8+ TCR transgenic CTLs, since the LCMV-8.7 variant virus (which is not recognized by transgenic TCR-expressing CTLs because of a point mutation) did not induce suppression. In addition, treatment with CD8 monoclonal antibody in vivo abrogated suppression. Once suppression had been established, it was found to be nonspecific. The abrogation of antibody responses depended on the relative kinetics of the antibody response involved and the kinetics of the anti-LCMV CTL response. Analysis of T- and B-cell subpopulations showed no significant changes, but immunohistochemical analysis of spleens revealed extensive destruction of follicular organization in lymphoid tissue by day 4 in transgenic mice infected with LCMV-WE but not in those infected with the CTL escape mutant LCMV-8.7. Impairment of antigen presentation rather than of T or B cells was also suggested by adoptive transfer experiments, showing that transferred infected macrophages may improve the anti-LCMV antibody response in LCMV-immunosuppressed transgenic recipients; also, T and B cells from suppressed transgenic mice did respond in irradiated and virus-infected nontransgenic mice with antibody formation to LCMV. Such virus-triggered, T-cell-mediated immunopathology causing the suppression of B cells and of protective antibody responses, including those against the infecting virus itself, may permit certain viruses to establish persistent infections.     

Agonistic anti-CD40 antibody profoundly suppresses the immune response to infection with lymphocytic choriomeningitis virus

Previous work has shown that agonistic Abs to CD40 (anti-CD40) can boost weak CD8 T cell responses as well as substitute for CD4 T cell function during chronic gammaherpes virus infection. Agonistic anti-CD40 treatment has, therefore, been suggested as a potential therapeutic strategy in immunocompromised patients. In this study, we investigated whether agonistic anti-CD40 could substitute for CD4 T cell help in generating a sustained CD8 T cell response and prevent viral recrudescence following infection with lymphocytic choriomeningitis virus (LCMV). Contrary to expectations, we found that anti-CD40 treatment of MHC class II-deficient mice infected with a moderate dose of LCMV resulted in severe suppression of the antiviral CD8 T cell response and uncontrolled virus spread, rather than improved CD8 T cell immune surveillance. In Ab-treated wild-type mice, the antiviral CD8 T cell response also collapsed prematurely, and virus clearance was delayed. Additional analysis revealed that, following anti-CD40 treatment, the virus-specific CD8 T cells initially proliferated normally, but an increased cell loss compared with that in untreated mice was observed. The anti-CD40-induced abortion of virus-specific CD8 T cells during LCMV infection was IL-12 independent, but depended partly on Fas expression. Notably, similar anti-CD40 treatment of vesicular stomatitis virus-infected mice resulted in an improved antiviral CD8 T cell response, demonstrating that the effect of anti-CD40 treatment varies with the virus infection studied. For this reason, we recommend further evaluation of the safety of this regimen before being applied to human patients.     

Long-Term CD4 Th1 and Th2 Memory following Acute Lymphocytic Choriomeningitis Virus Infection

CD4 T cells play a central role in viral immunity. They provide help for B cells and CD8 T cells and can act as effectors themselves. Despite their importance, relatively little is known about the magnitude and duration of virus-specific CD4 T-cell responses. In particular, it is not known whether both CD4 Th1 memory and CD4 Th2 memory can be induced by viral infections. To address these issues, we quantitated virus-specific CD4 Th1 (interleukin 2 [IL-2] and gamma-interferon) and Th2 (IL-4) responses in mice acutely infected with lymphocytic choriomeningitis virus (LCMV). Using two sensitive assays (enzyme-linked immunospot assay and intracellular stain) to measure cytokine production at the single-cell level, we found that both CD4 Th1 and Th2 responses were induced during primary LCMV infection. At the peak (day 8) of the response, the frequency of LCMV-specific CD4 Th1 cells was 1/35 to 1/160 CD4 T cells, and the frequency of Th2 cells was 1/400. After viral clearance, the numbers of virus-specific CD4 T cells dropped to 1/260 to 1/3,700 and then were maintained at this level indefinitely. Upon rechallenge with LCMV, both CD4 Th1 and Th2 memory cells made an anamnestic response in vivo. These results show that unlike some microbial infections in which only Th1 or Th2 responses are seen, an acute viral infection can induce a mixed CD4 T-cell response with long-term memory.     

Suppression by cyclosporin A of murine T-cell-mediated immunity against viruses in vivo and in vitro

The immunosuppressive effect of Cyclosporin A on T-cell-mediated antiviral immune responses was examined. When administered intraperitoneally CS-A abrogated anti-vaccinia virus, anti-lymphocytic choriomeningitis virus (LCMV), and anti-vesicular stomatitis virus (VSV) T-cell responses in a dose-dependent fashion. Usually 50-60 mg/kg were efficient in suppressing primary T-cell responses completely. In contrast, 10-20 mg/kg often enhanced T-cell responses significantly when compared with controls. Suppression was observed if CS-A treatment was started before virus injection and up to 12 hr after infection; CS-A given 24 hr after the virus still suppressed T-cell activity partially. A 50 mg/kg dose of CS-A suppressed secondary anti-vaccinia virus or anti-VSV T-cell responses in vivo by a factor of about 10. This dose suppressed the primary T-cell-dependent footpad swelling induced by local LCMV infection and prevented T-cell-mediated immunopathological death due to LCM when LCMV was injected intracerebrally. In addition, clearance of LCMV was delayed drastically by CS-A treatment. When added to cultures of in vivo-primed antiviral T cells that were restimulated in vitro, CS-A inhibited both proliferation as well as generation of virus-specific cytotoxic T cells in a dose-dependent way. The results show that in CS-A-treated mice primary and secondary antiviral T-cell responses are strongly inhibited; acute viral infections with cytopathic viruses may therefore be more dramatic. In contrast immunopathological T-cell-mediated disease caused by noncytopathic viruses such as LCMV may be prevented or attenuated.     

Intestinal intraepithelial lymphocytes exert potent protective cytotoxic activity during an acute virus infection

After systemic infection of mice with 104 PFU of lymphocytic choriomeningitis virus (LCMV), infected cells are detected simultaneously in various organs, including spleen and intestinal mucosa. Most notably, virus-infected cells are also present among CD11c+ dendritic cells in the subepithelial area of the small intestinal mucosa. Some of these virus-infected cells are in close spatial association with intestinal intraepithelial lymphocytes (IEL). Therefore, we compared virus-specific cytotoxic activity of CD8 splenocytes with that of IEL subsets. While ex vivo isolated TCRalphabeta+CD8alphaalpha+ IEL exert only minimal virus-specific cytotoxicity, maximum specific killing mediated by TCRalphabeta+CD8alphabeta+ IEL on day 8 postinfection exceeds maximum cytotoxic activity observed with CD8 splenocytes when assessed in vitro. Maximum cytotoxic activity of IEL is preceded by peak perforin and granzyme B mRNA expression in IEL around day 6 postinfection, suggesting a recent activation in situ. The antivirus cytotoxicity of in vivo primed IEL is further demonstrated by the protection from virus production in the spleen of mice infected with LCMV 10 h before adoptive cell transfer. These data indicate a potent priming of LCMV-specific IEL in situ after systemic LCMV infection and suggest that cytotoxic IEL markedly contribute to the elimination of virus-infected cells in the intestinal mucosa.