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

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

<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

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

<i>Emv2</i> preferentially selects against non-Vα2 env-specific CD4⁺ T cells.

<p>(A) Dose-response to env_124-138L stimulation of 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. (B) Frequency of env_124-138L-specific cells in Vα2 or non-Vα2 primary CD4⁺ T cells from the same donors. (C) Functional avidity of <i>Emv2</i>-selected (B6-EF4.1) or -nonselected (B6-EF4.1 <i>Emv2</i>^−/−) EF4.1 CD4⁺ T cells for env_124-138L. (D) Frequency of Vα2 cells in env_124-138L-specific CD4⁺ T cells from the same donors as a function of peptide concentration. (E) Frequency of env_124-138Y-specific cells in Vα2 or non-Vα2 primary CD4⁺ T cells from the same donors. (F) Functional avidity of <i>Emv2</i>-selected (B6-EF4.1) or -nonselected (B6-EF4.1 <i>Emv2</i>^−/−) EF4.1 CD4⁺ T cells for env_124-138Y. Numbers in (C) and (F) represent the ED₅₀. Data in (A–F) are the means ± SEM (<i>n</i> = 9–12) of 18-hr stimulations from 3 experiments.</p

<i>Emv2</i> selects against a fraction of env_124-138L-specific CD4⁺ T cells.

<p>(A) Dilution of prior CFSE label by primary EF4.1 CD4⁺ T cells incubated for three days <i>in vitro</i> in the absence of peptide stimulation (-) or in the presence of 10⁻⁵ M env_124-138L or env_124-138Y peptides. Numbers within the plots denote the percentage of CFSE⁻ cells and are representative of 4 mice per condition. (B) IL-2 production by three env_124-138L-specific hybridoma T cell lines in response to <i>in vitro</i> stimulation with the same peptides at 5×10⁻⁶ M. (C) <i>Emv2</i> transcription, relative to <i>Hprt</i> transcription, in thymi (Th) and spleens (Sp) of wild-type B6 or <i>Emv2</i>-deficient B6 mice (B6-<i>Emv2</i>^−/−). Each symbol is an individual mouse. The dashed line represents the limit of detection. (D) Frequency of env_124-138L-specific cells in primary CD4⁺ T cells from 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) from 3 experiments.</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