Annette Oxenius

HIV preferentially infects HIV-specific CD4+ T cells

HIV infection is associated with the progressive loss of CD4+ T cells through their destruction or decreased production. A central, yet unresolved issue of HIV disease is the mechanism for this loss, and in particular whether HIV-specific CD4+ T cells are preferentially affected. Here we show that HIV-specific memory CD4+ T cells in infected individuals contain more HIV viral DNA than other memory CD4+ T cells, at all stages of HIV disease. Additionally, following viral rebound during interruption of antiretroviral therapy, the frequency of HIV viral DNA in the HIV-specific pool of memory CD4+ T cells increases to a greater extent than in memory CD4+ T cells of other specificities. These findings show that HIV-specific CD4+ T cells are preferentially infected by HIV in vivo. This provides a potential mechanism to explain the loss of HIV-specific CD4+ T-cell responses, and consequently the loss of immunological control of HIV replication. Furthermore, the phenomenon of HIV specifically infecting the very cells that respond to it adds a cautionary note to the practice of structured therapy interruption.

Delay of HIV-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies

To determine the protective potential of the humoral immune response against HIV-1 in vivo we evaluated the potency of three neutralizing antibodies (2G12, 2F5 and 4E10) in suppressing viral rebound in six acutely and eight chronically HIV-1–infected individuals undergoing interruption of antiretroviral treatment (ART). Only two of eight chronically infected individuals showed evidence of a delay in viral rebound during the passive immunization. Rebound in antibody-treated acutely infected individuals upon cessation of ART was substantially later than in a control group of 12 individuals with acute infection. Escape mutant analysis showed that the activity of 2G12 was crucial for the in vivo effect of the neutralizing antibody cocktail. By providing further direct evidence of the potency, breadth and titers of neutralizing antibodies that are required for in vivo activity, these data underline both the potential and the limits of humoral immunity in controlling HIV-1 infection.

IL-21R on T Cells Is Critical for Sustained Functionality and Control of Chronic Viral Infection

Controlling Chronic Viral Infections Chronic viral infections such as HIV and hepatitis B and C viruses are major public health concerns. T cell—mediated immune responses are critical for controlling viral infections. In contrast to acute infections, chronic viral infections are characterized by “exhausted” cytotoxic CD8+ T cells, cells which exhibit reduced proliferative capacity, cytokine secretion, and cytotoxicity. Treatments that reverse exhaustion result in increased viral control. Despite their exhaustion, these CD8+ T cells eventually help to control chronic infections by killing virally infected cells, and require CD4+ T cell help to do so. How do CD4+ T cells provide help to CD8+ T cells during chronic infection (see the Perspective by Johnson and Jameson)? Elsaesser et al. (p. 1569, published online 7 May), Yi et al. (p. 1572, published online 14 May), and Fröhlich et al. (p. 1576, published online 28 May) now show that the cytokine, interleukin-21 (IL-21), known to be critical for the differentiation of certain CD4+ T cell effector subsets, is an essential factor produced by CD4+ T cells that helps CD8+ T cells to control chronic lymphocytic choriomeningitis virus infection in mice. Acute and chronic infections resulted in differing amounts of IL-21 production by virus-specific CD4+ T cells. CD8+ T cells required IL-21 directly, and when CD8+ T cells were unable to signal through IL-21 or IL-21 was not available, they were reduced in number, exhibited a more exhausted phenotype, and were not able to control the virus. In contrast, the absence of IL-21–dependent signaling did not affect primary CD8+ T cell responses to acute infection or responses to a viral rechallenge, suggesting that differentiation of memory CD8+ T cells is independent of IL-21. Thymus cells of the immune system require the cytokine interleukin-21 to control chronic viral infections. Chronic viral infection is often associated with the dysfunction of virus-specific T cells. Our studies using Il21r-deficient (Il21r–/–) mice now suggest that interleukin-21 (IL-21) is critical for the long-term maintenance and functionality of CD8+ T cells and the control of chronic lymphocytic choriomeningitis virus infection in mice. Cell-autonomous IL-21 receptor (IL-21R)–dependent signaling by CD8+ T cells was required for sustained cell proliferation and cytokine production during chronic infection. Il21r–/– mice showed normal CD8+ T cell expansion, effector function, memory homeostasis, and recall responses during acute and after resolved infection with several other nonpersistent viruses. These data suggest that IL-21R signaling is required for the maintenance of polyfunctional T cells during chronic viral infections and have implications for understanding the immune response to other persisting antigens, such as tumors.

Virus-specific major MHC class II-restricted TCR-transgenic mice: effects on humoral and cellular immune responses after viral infection

A transgenic mouse expressing MHC class II‐restricted TCR with specificity for a lymphocytic choriomeningitis virus (LCMV) glycoprotein‐derived T helper cell epitope was developed to study the role of LCMV‐specific CD4+ T cells in virus infection in vivo. The majority of CD4+ T cells in TCR transgenic mice expressed the transgenic receptor, and LCMV glycoprotein‐specific TCR transgenic CD4+ T cells efficiently mediated help for the production of LCMV glycoprotein‐specific isotype‐switched antibodies. In contrast, LCMV glycoprotein‐specific TCR transgenic mice exhibited a drastically reduced ability to provide help for the generation of antibody responses specific for the virus‐internal nucleoprotein, indicating that intramolecular/intrastructural help is limited to antigens that are accessible to B cells on the viral surface. Antiviral cellular immunity was studied with noncytopathic LCMV and recombinant cytopathic vaccinia virus expressing the LCMV glycoprotein. TCR transgenic mice failed to efficiently control LCMV infection, demonstrating that functional LCMV‐specific CD4+ T cells – even if activated and present at extremely high frequencies – cannot directly mediate protective immunity against LCMV. Despite the fact that LCMV‐primed CD4+ T cells from TCR transgenic mice as well as from control mice showed low MHC class II‐restricted cytotoxic activity in vivo, this did not correlate with protection against LCMV replication in vivo. In contrast, CD4+ T cells from TCR‐transgenic mice mediated efficient protection against infection with recombinant vaccinia virus. These results further support the need for different immune effector functions for protective immunity against different viral infections.

Disseminated and sustained HIV infection in CD34+ cord blood cell-transplanted Rag2-/-gamma c-/- mice.

Because of species selectivity, HIV research is largely restricted to in vitro or clinical studies, both limited in their ability to rapidly assess new strategies to fight the virus. To prospectively study some aspects of HIV in vivo, immunodeficient mice, transplanted with either human peripheral blood leukocytes or human fetal tissues, have been developed. Although these are susceptible to HIV infection, xenoreactivity, and short infection spans, resource and ethical constraints, as well as biased HIV coreceptor tropic strain infection, pose substantial problems in their use. Rag2−/−γc−/− mice, transplanted as newborns with human CD34+ cells, were recently shown to develop human B, T, and dendritic cells, constituting lymphoid organs in situ. Here we tested these mice as a model system for HIV-1 infection. HIV RNA levels peaked to up to 2 × 106 copies per milliliter of plasma early after infection, and viremia was observed for up to 190 days, the longest time followed. A marked relative CD4+ T cell depletion in peripheral blood occurred in CXCR4-tropic strain-infected mice, whereas this was less pronounced in CCR5-tropic strain-infected animals. Thymus infection was almost exclusively observed in CXCR4-tropic strain-infected mice, whereas spleen and lymph node HIV infection occurred irrespective of coreceptor selectivity, consistent with respective coreceptor expression on human CD4+ T cells. Thus, this straightforward to generate and cost-effective in vivo model closely resembles HIV infection in man and therefore should be valuable to study virus-induced pathology and to rapidly evaluate new approaches aiming to prevent or treat HIV infection.

Functional Properties and Lineage Relationship of CD8+ T Cell Subsets Identified by Expression of IL-7 Receptor α and CD62L

Three major subsets of Ag-experienced CD8+ T cells have been identified according to their expression of CD62L and CD127. These markers are associated with central memory T cells (CD62L+CD127+), effector memory T cells (CD162L−CD127+), and effector T cells (CD62L−CD127−). In this study we characterized the development of these three populations during acute and chronic viral infections and after immunization with virus-like particles and determined their lineage relation and functional and protective properties. We found that the balance between the three subsets was critically regulated by the availability of Ag and time. After initial down-regulation of CD127, the responding CD8+ T cell population down-regulated CD62L and re-expressed CD127. Dependent on Ag availability, the cells then further differentiated into CD62L−CD127− effector cells or, in the absence of Ag, re-expressed CD62L to become central memory T cells. Although all three populations efficiently produced effector cytokines such as IFN-γ, CD62L−CD127− effector cells exhibited the highest ex vivo lytic potential. In contrast, CD62L+CD127+ central memory T cells most efficiently produced IL-2 and proliferated extensively in vitro and in vivo upon antigenic restimulation. Strikingly, only effector and effector memory, but not central memory, T cells were able to protect against peripheral infection with vaccinia virus, whereas central memory T cells were most potent at protecting against systemic infection with lymphocytic choriomeningitis virus, indicating that the antiviral protective capacities of specific CD8+ T cell subsets are closely related to the nature of the challenging pathogen.

Immediate Cytotoxicity But Not Degranulation Distinguishes Effector and Memory Subsets of CD8+ T Cells

CD8+ T cells play a central role in the resolution and containment of viral infections. A key effector function of CD8+ T cells is their cytolytic activity toward infected cells. Here, we studied the regulation of cytolytic activity in naive, effector, and central versus effector memory CD8+ T cells specific for the same glycoprotein-derived epitope of lymphocytic choriomeningitis virus. Our results show that the kinetics of degranulation, assessed by a novel flow cytometric based assay, were identical in effector and both subsets of memory CD8+ T cells, but absent in naive CD8+ T cells. However, immediate cytolytic activity was most pronounced in effector T cells, low in effector memory T cells, and absent in central memory T cells, correlating with the respective levels of cytolytic effector molecules present in lytic granules. These results indicate that an inherent program of degranulation is a feature of antigen-experienced cells as opposed to naive CD8+ T cells and that the ability of CD8+ T cells to induce target cell apoptosis/death is dependent on granule protein content rather than on the act of degranulation itself. Furthermore, these results provide a potential mechanism by which central memory CD8+ T cell–mediated death of antigen-presenting cells within the lymph node is avoided.

Stimulation of HIV-specific cellular immunity by structured treatment interruption fails to enhance viral control in chronic HIV infection

Potent antiretroviral therapy (ART) suppresses HIV-1 viral replication and results in decreased morbidity and mortality. However, prolonged treatment is associated with drug-induced toxicity, emergence of drug-resistant viral strains, and financial constraints. Structured therapeutic interruptions (STIs) have been proposed as a strategy that could boost HIV-specific immunity, through controlled exposure to autologous virus over limited time periods, and subsequently control viral replication in the absence of ART. Here, we analyzed the impact of repeated STIs on virological and immunological parameters in a large prospective STI study. We show that: (i) the plateau virus load (VL) reached after STIs correlated with pretreatment VL, the amount of viral recrudescence during the treatment interruptions, and the off-treatment viral rebound rate; (ii) the magnitude and the breadth of the HIV-specific CD8+ T lymphocyte response, despite marked interpatient variability, increased overall with STI. However, the quantity and quality of the post-STI response was comparable to the response observed before any therapy; (iii) individuals with strong and broad HIV-specific CD8+ T lymphocyte responses at baseline retained these characteristics during and after STI; (iv) the increase in HIV-specific CD8+ T lymphocyte frequencies induced by STI was not correlated with decreased viral set point after STI; and (v) HIV-specific CD4+ T lymphocyte responses increased with STI, but were subsequently maintained only in patients with low pretreatment and plateau VLs. Overall, these data indicate that STI-induced quantitative boosting of HIV-specific cellular immunity was not associated with substantial change in viral replication and that STI was largely restoring pretherapy CD8+ T cell responses in patients with established infection.

COMPARISON OF ACTIVATION VERSUS INDUCTION OF UNRESPONSIVENESS OF VIRUS-SPECIFIC CD4+ AND CD8+ T CELLS UPON ACUTE VERSUS PERSISTENT VIRAL INFECTION

The functional status of CD4+ T cells during establishment of persistent infection with the noncytopathic lymphocytic choriomeningitis virus was assessed and compared to that of cytotoxic CD8+ T cells. Functionality of virus-specific CD4+ T cells was measured by proliferative responses, cytokine secretion, cognate help, and IFNgamma-mediated protection against challenge infection with recombinant vaccinia virus. Functional CD4+ T cells were induced early after infection and remained measurable up to 6 weeks but then were rendered unresponsive. In contrast, CD8+ T cells were functionally inactivated within 10-15 days. Importantly, functional inactivation of virus-specific CD4+ T cells during persistent viral infection seemed to be critical for the survival of the host.

Differential role of IL-2R signaling for CD8+ T cell responses in acute and chronic viral infections

IL‐2 is a cytokine with multiple and even divergent functions; it has been described as a key cytokine for in vitro T cell proliferation but is also essential for down‐regulating T cell responses by inducing activation‐induced cell death as well as regulatory T cells. The in vivo analysis of IL‐2 function in regulating specific T cell responses has been hampered by the fact that mice deficient in IL‐2 or its receptors develop lymphoproliferative diseases and/or autoimmunity. Here we generated chimeric mice harboring both IL‐2R‐competent and IL‐2R‐deficient T cells and assessed CD8+ T cell induction, function and maintenance after acute or persistent viral infections. Induction and maintenance of CD8+ T cells were relatively independent of IL‐2R signaling during acute/resolved viral infection. In marked contrast, IL‐2 was crucial for secondary expansion of memory CD8+ T cells and for the maintenance of virus‐specific CD8+ T cells during persistent viral infections. Thus, depending on the chronicity of antigen exposure, IL‐2R signaling is either essential or largely dispensable for induction and maintenance of virus‐specific CD8+ T cell responses.