Negative Selection by an Endogenous Retrovirus Promotes a Higher-Avidity CD4+ T Cell Response to Retroviral Infection

Effective T cell responses can decisively influence the outcome of retroviral infection. However, what constitutes protective T cell responses or determines the ability of the host to mount such responses is incompletely understood. Here we studied the requirements for development and induction of CD4+ T cells that were essential for immunity to Friend virus (FV) infection of mice, according to their TCR avidity for an FV-derived epitope. We showed that a self peptide, encoded by an endogenous retrovirus, negatively selected a significant fraction of polyclonal FV-specific CD4+ T cells and diminished the response to FV infection. Surprisingly, however, CD4+ T cell-mediated antiviral activity was fully preserved. Detailed repertoire analysis revealed that clones with low avidity for FV-derived peptides were more cross-reactive with self peptides and were consequently preferentially deleted. Negative selection of low-avidity FV-reactive CD4+ T cells was responsible for the dominance of high-avidity clones in the response to FV infection, suggesting that protection against the primary infecting virus was mediated exclusively by high-avidity CD4+ T cells. Thus, although negative selection reduced the size and cross-reactivity of the available FV-reactive naïve CD4+ T cell repertoire, it increased the overall avidity of the repertoire that responded to infection. These findings demonstrate that self proteins expressed by replication-defective endogenous retroviruses can heavily influence the formation of the TCR repertoire reactive with exogenous retroviruses and determine the avidity of the response to retroviral infection. Given the overabundance of endogenous retroviruses in the human genome, these findings also suggest that endogenous retroviral proteins, presented by products of highly polymorphic HLA alleles, may shape the human TCR repertoire that reacts with exogenous retroviruses or other infecting pathogens, leading to interindividual heterogeneity

Distinct expression profiles of TGF-β1 signaling mediators in pathogenic SIVmac and non-pathogenic SIVagm infections

BACKGROUND: The generalized T-cell activation characterizing HIV-1 and SIVmac infections in humans and macaques (MACs) is not found in the non-pathogenic SIVagm infection in African green monkeys (AGMs). We have previously shown that TGF-β1, Foxp3 and IL-10 are induced very early after SIVagm infection. In SIVmac-infected MACs, plasma TGF-β1 induction persists during primary infection [1]. We raised the hypothesis that MACs are unable to respond to TGF-β1 and thus cannot resorb virus-driven inflammation. We therefore compared the very early expression dynamics of pro- and anti-inflammatory markers as well as of factors involved in the TGF-β1 signaling pathway in SIV-infected AGMs and MACs. METHODS: Levels of transcripts encoding for pro- and anti-inflammatory markers (tnf-α, ifn-γ, il-10, t-bet, gata-3) as well as for TGF-β1 signaling mediators (smad3, smad4, smad7) were followed by real time PCR in a prospective study enrolling 6 AGMs and 6 MACs. RESULTS: During primary SIVmac infection, up-regulations of tnf-α, ifn-γ and t-bet responses (days 1–16 p.i.) were stronger whereas il-10 response was delayed (4(th )week p.i.) compared to SIVagm infection. Up-regulation of smad7 (days 3–8 p.i.), a cellular mediator inhibiting the TGF-β1 signaling cascade, characterized SIV-infected MACs. In AGMs, we found increases of gata-3 but not t-bet, a longer lasting up-regulation of smad4 (days 1–21 p.i), a mediator enhancing TGF-β1 signaling, and no smad7 up-regulations. CONCLUSION: Our data suggest that the inability to resorb virus-driven inflammation and activation during the pathogenic HIV-1/SIVmac infections is associated with an unresponsiveness to TGF-β1

Race between Retroviral Spread and CD4+ T-Cell Response Determines the Outcome of Acute Friend Virus Infection▿

Retroviruses can establish persistent infection despite induction of a multipartite antiviral immune response. Whether collective failure of all parts of the immune response or selective deficiency in one crucial part underlies the inability of the host to clear retroviral infections is currently uncertain. We examine here the contribution of virus-specific CD4+ T cells in resistance against Friend virus (FV) infection in the murine host. We show that the magnitude and duration of the FV-specific CD4+ T-cell response is directly proportional to resistance against acute FV infection and subsequent disease. Notably, significant protection against FV-induced disease is afforded by FV-specific CD4+ T cells in the absence of a virus-specific CD8+ T-cell or B-cell response. Enhanced spread of FV infection in hosts with increased genetic susceptibility or coinfection with Lactate dehydrogenase-elevating virus (LDV) causes a proportional increase in the number of FV-specific CD4+ T cells required to control FV-induced disease. Furthermore, ultimate failure of FV/LDV coinfected hosts to control FV-induced disease is accompanied by accelerated contraction of the FV-specific CD4+ T-cell response. Conversely, an increased frequency or continuous supply of FV-specific CD4+ T cells is both necessary and sufficient to effectively contain acute infection and prevent disease, even in the presence of coinfection. Thus, these results suggest that FV-specific CD4+ T cells provide significant direct protection against acute FV infection, the extent of which critically depends on the ratio of FV-infected cells to FV-specific CD4+ T cells

Modulation of Type I interferon-associated viral sensing during acute simian immunodefiency virus (SIV) infection in African green monkeys Running title: Very early effects of SIV infection on viral sensing

International audienceNatural hosts of simian immunodeficiency virus (SIV), such as African green monkeys (AGMs), do not progress to AIDS when infected with SIV. This is associated with an absence of a chronic type I interferon (IFN-I) signature. It is unclear how the IFN-I response is downmodulated in AGMs. We longitudinally assessed the capacity of AGM blood cells to produce IFN-I in response to SIV and herpes simplex virus (HSV) infection. Phenotypes and functions of plasmacytoid dendritic cells (pDCs) and other mononuclear blood cells were assessed by flow cytometry, and expression of viral sensors was measured by reverse transcription-PCR. pDCs displayed low BDCA-2, CD40, and HLA-DR expression levels during AGM acute SIV (SIVagm) infection. BDCA-2 was required for sensing of SIV, but not of HSV, by pDCs. In acute infection, AGM peripheral blood mononuclear cells (PBMCs) produced less IFN-I upon SIV stimulation. In the chronic phase, the production was normal, confirming that the lack of chronic inflammation is not due to a sensing defect of pDCs. In contrast to stimulation by SIV, more IFN-I was produced upon HSV stimulation of PBMCs isolated during acute infection, while the frequency of AGM pDCs producing IFN-I upon in vitro stimulation with HSV was diminished. Indeed, other cells started producing IFN-I. This increased viral sensing by non-pDCs was associated with an upregulation of Toll-like receptor 3 and IFN-γ-inducible protein 16 caused by IFN-I in acute SIVagm infection. Our results suggest that, as in pathogenic SIVmac infection, SIVagm infection mobilizes bone marrow precursor pDCs. Moreover, we show that SIV infection modifies the capacity of viral sensing in cells other than pDCs, which could drive IFN-I production in specific settings.IMPORTANCE:The effects of HIV/SIV infections on the capacity of plasmacytoid dendritic cells (pDCs) to produce IFN-I in vivo are still incompletely defined. As IFN-I can restrict viral replication, contribute to inflammation, and influence immune responses, alteration of this capacity could impact the viral reservoir size. We observed that even in nonpathogenic SIV infection, the frequency of pDCs capable of efficiently sensing SIV and producing IFN-I was reduced during acute infection. We discovered that, concomitantly, cells other than pDCs had increased abilities for viral sensing. Our results suggest that pDC-produced IFN-I upregulates viral sensors in bystander cells, the latter gaining the capacity to produce IFN-I. These results indicate that in certain settings, cells other than pDCs can drive IFN-I-associated inflammation in SIV infection. This has important implications for the understanding of persistent inflammation and the establishment of viral reservoirs

<i>Emv2</i>-selected CD4⁺ T cells mount a predominantly high-avidity response.

<p>(A–C) CD45.2⁺ (<i>Ptprc</i>^2/2) CD4⁺ T cells isolated from either B6 (B6-EF4.1) or <i>Emv2</i>-deficient B6 (B6-EF4.1 <i>Emv2</i>^−/−) EF4.1 donor mice were adoptively transferred into <i>Ptprc</i>^1/2 B6 recipients that were infected with FV the same day and analyzed 7 days later. (A) Absolute number of total, Vα2 or non-Vα2 FV-responding (CD44^hi) donor (CD45.2⁺CD45.1⁻) CD4⁺ T cells isolated from the spleens of recipient mice according to donor type. (B) Flow cytometric example and (C) frequency of high-avidity Vα2 cells in responding CD4⁺ T cells according to donor type. In (A) and (C) each symbol is an individual mouse.</p

Detection of env-specific CD4⁺ T cells by A^b-env_123-141 tetramer eclipsed by antigen-induced TCR downregulation.

<p>(A) A^b-hCLIP (control) or A^b-env_123-141 tetramer staining in total CD4⁺ T cells isolated from the spleen of wild-type B6 mice 7 days post FV infection. Plots are representative of 7 mice. (B) Frequency of Vα2 cells in either bulk naïve (CD44^lo), bulk memory (CD44^hi) or A^b- env_123-141 tetramer⁺ CD4⁺ T cells from the same FV infected mice. Horizontal short lines in naïve and memory subsets denote the mean frequency of Vα2 cells in the same populations from uninfected mice. Each symbol represents an individual mouse. (C–F) CD45.1⁺ EF4.1 CD4⁺ T cells were adoptively transferred into wild-type B6 recipients that were infected with FV the same day. (C) A^b-env_123-141 tetramer staining in host (CD45.1⁻) or donor (CD45.1⁺) CD4⁺ T cells according to TCRα or TCRβ staining. Gates in donor CD4⁺ T cells are set around the median TCRα and TCRβ staining, respectively. (D) Percentage of A^b-env_123-141 tetramer⁺ cells in donor CD4⁺ T cells with TCRβ (<i>left</i>) or TCRα (<i>right</i>) staining below or above the median. (E) A^b-hCLIP or A^b-env_123-141 tetramer staining in host or donor CD4⁺ T cells from the same recipients assessed directly <i>ex vivo</i> (<i>top</i>) or following 3-day <i>in vitro</i> culture in the absence of antigenic stimulation (<i>bottom</i>). (F) Percentage of A^b-env_123-141 tetramer⁺ cells in donor CD4⁺ T cells before and after <i>in vitro</i> culture. In (D) and (F) each symbol represents an individual mouse from one of two experiments.</p

Depth of Vα2 or non-Vα2 env-specific CD4⁺ T cell repertoires.

<p>(A–C) Vα2 or non-Vα env_124-138L-reactive hybridoma T cell lines were derived from <i>Emv2</i>^+/+ (B6-EF4.1) or <i>Emv2</i>^−/− (B6-EF4.1 <i>Emv2</i>^−/−) EF4.1 mice and tested for reactivity against a library of env_126-138 peptide epitopes. The amino acid residues in positions 128 (A), 129 (B) and 133 (C) that elicited at least 40% of the maximal response are listed in the order of preference by the individual clones.</p

Cross-reactivity of individual Vα2 or non-Vα2 CD4⁺ T cells.

<p>(A) Frequency of env_124-138YS-reactive cells in Vα2 or non-Vα2 CD4⁺ T cells isolated from either B6 (B6-EF4.1) or <i>Emv2</i>-deficient B6 (B6-EF4.1 <i>Emv2</i>^−/−) EF4.1 mice. Data are the means ± SEM (<i>n</i> = 9) of 18-hr stimulations from 3 experiments. (B–C) IL-2 production in response to stimulation with 5×10⁻⁶ M env_124-138L (L), env_124-138Y (Y) or env_124-138YS (YS) in comparison with the absence of peptide stimulation (-) of Vα2 or non-Vα2 env_124-138L-reactive hybridoma T cell lines derived from <i>Emv2</i>^+/+ (B) or <i>Emv2</i>^−/− (C) EF4.1 mice.</p

<i>Emv2</i>-selected CD4⁺ T cells retain full antiviral activity.

<p>(A) Mean frequency (± SEM, <i>n</i> = 8–19) of FV-infected (glyco-Gag⁺) Ter119⁺ cells in the spleens of FV-infected B6 or <i>Emv2</i>-deficient B6 mice (B6-<i>Emv2</i>^−/−). (B–C) CD4⁺ T cells isolated from either B6 (B6-EF4.1) or <i>Emv2</i>-deficient B6 (B6-EF4.1 <i>Emv2</i>^−/−) EF4.1 mice were adoptively transferred into B6 or B6.A-<i>Fv2</i>^s recipients that were infected with FV the same day and analyzed 7 days later. (B) Flow cytometric example of FV-infected Ter119⁺ cells from B6 recipients and (C) frequency of FV-infected cells in Ter119⁺ cells from the spleens of B6 or B6.A-<i>Fv2</i>^s recipients of CD4⁺ T cells. Control B6 and B6.A-<i>Fv2</i>^s mice that received no CD4⁺ T cells (-) are also included. Each symbol is an individual mouse. (D) Spleen index (<i>left</i>) and RBC count (<i>right</i>) of B6-<i>Rag1</i>^−/−<i>Fv2</i>^s mice that were infected with FV and either received the same day CD4⁺ T cells isolated from either B6 (B6-EF4.1) or <i>Emv2</i>-deficient B6 (B6-EF4.1 <i>Emv2</i>^−/−) EF4.1 mice or no cells (-). Each symbol is an individual mouse analyzed 3 weeks post infection. (E) Titers of FV-neutralizing antibodies during the course of FV infection (<i>left</i>) and titers of F-MLV-infected cell-binding IgG (<i>middle</i>) and IgM (<i>right</i>) 7 days post FV infection, in the sera of B6-<i>Tcra</i>^−/− mice that either received CD4⁺ T cells isolated from either B6 (B6-EF4.1) or <i>Emv2</i>-deficient B6 (B6-EF4.1 <i>Emv2</i>^−/−) EF4.1 mice or no cells (-) the day of the infection. Dashed lines represent the limit of detection. Data are the means ± SEM (<i>n</i> = 11–12) from 2 experiments.</p

Genetic contribution to a high-avidity env-reactive CD4⁺ T cell repertoire.

<p>(A) Frequency of env_124-138L- reactive cells in Vα2 or non-Vα2 primary CD4⁺ T cells isolated from either B6 (B6-EF4.1) or 129S8 (129S8-EF4.1) EF4.1 mice. (B) Functional avidity of env_124-138L-reactive Vα2 or non-Vα2 primary CD4⁺ T cells from the same donors in A. (C) Frequency of Vα2 cells in env_124-138L-reactive CD4⁺ T cells from the same donors in A as a function of peptide concentration. (D) Frequency of env_124-138L- reactive cells in Vα2 or non-Vα2 primary CD4⁺ T cells isolated from either B6×129S8-EF4.1 F₁, B6-<i>Emv2</i>^−/−×129S8-EF4.1 F₁ or B6-<i>Tcra</i>^−/−×129S8-EF4.1 F₁, EF4.1 mice. (E) Functional avidity of env_124-138L-reactive Vα2 or non-Vα2 primary CD4⁺ T cells from the same donors in D. (F) Frequency of Vα2 cells in env_124-138L-reactive CD4⁺ T cells from the same donors in D as a function of peptide concentration. Numbers in (B) and (E) represent the ED₅₀. In (C) and (F) the CD4⁺ T cell response elicited by the last peptide dose (10⁻⁸ M) was too small to allow accurate measurement of the frequency of Va2 cells and was therefore omitted. Data in (A–F) are the means ± SEM (<i>n</i> = 4–8) of 18-hr stimulations from 3 experiments.</p