Inefficient Nef-Mediated Downmodulation of CD3 and MHC-I Correlates with Loss of CD4+ T Cells in Natural SIV Infection

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Abstract
Recent data suggest that Nef-mediated downmodulation of TCR-CD3 may protect SIVsmm-infected sooty mangabeys (SMs) against the loss of CD4+ T cells. However, the mechanisms underlying this protective effect remain unclear. To further assess the role of Nef in nonpathogenic SIV infection, we cloned nef alleles from 11 SIVsmm-infected SMs with high (>500) and 15 animals with low (+ T-cells/µl in bulk into proviral HIV-1 IRES/eGFP constructs and analyzed their effects on the phenotype, activation, and apoptosis of primary T cells. We found that not only efficient Nef-mediated downmodulation of TCR-CD3 but also of MHC-I correlated with preserved CD4+ T cell counts, as well as with high numbers of Ki67+CD4+ and CD8+CD28+ T cells and reduced CD95 expression by CD4+ T cells. Moreover, effective MHC-I downregulation correlated with low proportions of effector and high percentages of naïve and memory CD8+ T cells. We found that T cells infected with viruses expressing Nef alleles from the CD4low SM group expressed significantly higher levels of the CD69, interleukin (IL)-2 and programmed death (PD)-1 receptors than those expressing Nefs from the CD4high group. SIVsmm Nef alleles that were less active in downmodulating TCR-CD3 were also less potent in suppressing the activation of virally infected T cells and subsequent cell death. However, only nef alleles from a single animal with very low CD4+ T cell counts rendered T cells hyper-responsive to activation, similar to those of HIV-1. Our data suggest that Nef may protect the natural hosts of SIV against the loss of CD4+ T cells by at least two mechanisms: (i) downmodulation of TCR-CD3 to prevent activation-induced cell death and to suppress the induction of PD-1 that may impair T cell function and survival, and (ii) downmodulation of MHC-I to reduce CTL lysis of virally infected CD4+ T cells and/or bystander CD8+ T cell activation. The accessory Nef protein is commonly considered a “pathogenicity” factor of primate lentiviruses. However, SIVs do not cause disease in their natural hosts, although they all encode nef genes and sustain high levels of viremia. To better understand the role of Nef in natural nonpathogenic SIV infection, we compared the function of Nef alleles from two groups of SIVsmm-infected sooty mangabeys: (i) those that maintained normal CD4+ T cell counts and (ii) a small subset (10%–15%) of animals that exhibited a considerable loss of CD4+ helper T cells. We found that the efficiency of two specific Nef functions, i.e., downmodulation of TCR-CD3 and MHC-I, correlated with preserved CD4+ T cell homeostasis, as well as with other immunological features, such as high numbers of proliferating CD4+ Ki67+ T cells. Moreover, lack of CD3 surface expression was associated with low levels of apoptosis and PD-1 expression by virally infected T cells. Thus, the ability of Nef to remove TCR-CD3 and MHC-I from the cell surface may help the natural hosts of SIV to maintain normal CD4+ T cell counts despite high levels of viral replication by preventing activation-induced cell death and CTL lysis of infected T cells and/or CD8+ T cell activation.