Regulatory T cells constrain the TCR repertoire of antigen-stimulated conventional CD4 T cells

Abstract

To analyze the potential role of Tregs in controlling the TCR repertoire breadth to a non-self-antigen, a TCRβ transgenic mouse model (EF4.1) expressing a limited, yet polyclonal naïve T-cell repertoire was used. The response of EF4.1 mice to an I-Ab-associated epitope of the F-MuLV envelope protein is dominated by clones expressing a Vα2 gene segment, thus allowing a comprehensive analysis of the TCRα repertoire in a relatively large cohort of mice. Control and Treg-depleted EF4.1 mice were immunized, and the extent of the Vα2-bearing, antigen-specific TCR repertoire was characterized by high-throughput sequencing and spectratyping analysis. In addition to increased clonal expansion and acquisition of effector functions, Treg depletion led to the expression of a more diverse TCR repertoire comprising several private clonotypes rarely observed in control mice or in the pre-immune repertoire. Injection of anti-CD86 antibodies in vivo led to a strong reduction in TCR diversity, suggesting that Tregs may influence TCR repertoire diversity by modulating costimulatory molecule availability. Collectively, these studies illustrate an additional mechanism whereby Tregs control the immune response to non-self-antigens.

Keywords: CD4+ T‐cell lymphocytes; TCR repertoire analysis; regulatory T cells.

Figure EV1. Increased env_122–141 peptide reactivity among Vα2‐expressing CD4⁺ T cells from EF4.1 TCRβ transgenic mice

Purified CD4⁺ T cells from naïve TCRβ‐tg mice were stimulated in vitro with splenic dendritic cells and graded doses of env_122–141 peptide, and analyzed for CD40L and CD69 expression 18 h later.

1. A, B

   Representative FACS contour plots of cells restimulated in control medium or in the presence of 10⁶ M env_122–141 peptide and mean percentage of CD69‐ and CD40L‐expressing cells in gated Vα2⁺ or Vα2⁻ CD4⁺ T cells. Symbols represent mean values (± s.e.m., n = 2 mice). Results are from three independent experiments.

Figure 1. Expansion and repertoire restriction of CD4⁺ Vα2⁺ T cells in EF4.1 TCRβ‐tg mice upon antigen stimulation

TCRβ‐tg mice were left untreated (NI) or immunized with env_122–141 peptide‐pulsed splenic dendritic cells (DC).

1. Representative FACS contour plots (left panel) and mean percentage of Ki67 expression (right panel) in gated CD4⁺ Vα2⁺ or Vα2⁻ T cells 5 days after immunization (+env) or not (NI) (n = 13–15 mice/group, Mann–Whitney test).

2. Representative FACS contour plots (left panel) and mean percentage of IFN‐γ expression (right panel) of in vivo primed, in vitro‐restimulated (10⁻⁶ M env_122–141 peptide) Vα2⁺ or Vα2⁻ CD4⁺ T cells (n = 14 mice/group, Mann–Whitney test).

3. Spectratyping plots of Vα2⁺ CD4⁺ T cells from C57BL/6 wild‐type, TCRβ‐tg‐naïve, immunized TCRβ‐tg, OTII, and Marilyn mice. Data are representative of four mice.

Data information: In both (A) and (B), histograms represent mean values (± s.e.m.) while each symbol refers to an individual mouse. Results are from four independent experiments.

Figure 2. Partial depletion of FoxP3⁺ regulatory T cells leads to an enhanced immune response to antigen

Control IgG‐ or anti‐CD25‐treated mice were immunized or not by in vivo transfer of env_122–141 peptide‐pulsed splenic dendritic cells (DC). At day 5 after immunization, the draining lymph nodes from immunized (env‐DC) and non‐immunized (NI) mice were harvested and analyzed by flow cytometry.

1. A, B

   Representative FACS contour plots and mean percentage of FoxP3‐expressing CD4⁺ T cells (n = 24 mice/group for immunized mice and 4 mice/group for NI mice, Mann–Whitney test).

2. C, D

   Representative FACS contour plots and mean percentage of Ki67 expression in gated Vα2⁺ or Vα2⁻ CD4⁺ T cells (n = 25–27 mice/group for immunized mice and four mice/group for NI mice, Student's t‐test).

Data information: In (B) and (D), histograms represent mean values (± s.e.m.) with symbols referring to an individual mouse. Results are from six independent experiments for immunized mice and one experiment for non‐immunized mice.

Figure 3. Treg depletion leads to enhanced cytokine production and functional avidity of Vα2⁺ antigen‐reactive CD4⁺ T cells

CD4⁺ T cells from the draining lymph nodes of IgG‐ or anti‐CD25‐treated TCRβ‐tg mice previously immunized with env_122–141 peptide‐pulsed DC were restimulated in vitro for 48 h with the same env_122–141 peptide.

1. Representative FACS contour plots of IFN‐γ‐expressing cells from control or env‐supplemented (10⁻⁶ M) cultures.

2. Mean percentage of IFN‐γ‐expressing cells in gated Vα2⁺ or Vα2⁻ CD4⁺ T cells.

3. Functional avidity, as measured by the percent maximal number of IFN‐γ‐expressing cells in gated Vα2⁺ CD4⁺ T cells.

4. Effective peptide concentration required to induce a half‐maximal response (EC₅₀).

Data information: In (B) and (C), symbols represent mean values (± s.e.m., n = 9 mice/group). Results are from two independent experiments. In (D), histograms represent mean values (± s.e.m., n = 7–8 mice/group, Mann–Whitney test) with each symbol referring to an individual mouse. Results are from two independent experiments.

Figure 4. Increased diversity of the CDR3Vα2 antigen‐reactive repertoire following Treg depletion

1. Spectratyping plots of immune CD4⁺ Vα2⁺ T cells derived from IgG (upper row) or anti‐CD25‐treated mice (see legend to Fig 3 for detailed protocol).

2. Index of oligoclonality for CDR3Vα2, determined as described in Materials and Methods section. Histograms represent mean values (± s.e.m., n = 17 mice/group for immunized mice and n = 4–7 mice for naïve wild‐type and TCRβ‐tg mice, Mann–Whitney test) with each symbol referring to an individual mouse. Results are from five independent experiments for immunized mice and from one experiment for naïve wild‐type and TCRβ‐tg mice.

Figure 5. High‐throughput TCR Vα2 sequencing reveals a skewed repertoire in response to antigen stimulation

TCRβ‐tg mice were immunized on one flank with env_122–141 peptide‐pulsed DC. Five days later, CD4⁺ T cells from non‐draining lymph nodes (LN) were collected for naïve repertoire analysis, while cells from the draining LN were restimulated for 48 h in vitro as described in the legend to Fig 3.

1. A, B

   Sum of frequencies and relative frequencies of the 10 most dominant TCR Vα2 nucleotide sequences.

2. C

   Lorenz curves representing the degree of skewness of the Vα2 TCR repertoire of CD4⁺ T cells from immune and non‐immune cells are displayed by plotting the cumulative frequency of unique sequences vs. the cumulative frequency of total sequences. The line of equality represents an even distribution of clonotypes.

3. D

   Gini coefficient was calculated for each group.

4. E

   Shannon diversity index was used to estimate clonal diversity and distribution.

Data information: In (A, B, D, and E), histograms represent mean values (± s.e.m., n = 4 mice, Mann–Whitney test) while each symbol refers to an individual mouse. In (C), the symbols represent mean values (± s.e.m., n = 4 mice).

Figure EV2. EF4.1 TCRβ‐tg mice display a polyclonal TCR Vα2 repertoire (related to Fig 6)

The CDR3α2 repertoire of purified CD4⁺ T cells from C57BL/6 wild‐type, TCRβ‐tg‐naïve, and TCRβ‐tg mice previously (day −4) treated with IgG or anti‐CD25 mAbs was analyzed by high‐throughput sequencing.

1. A

   Sum of frequencies of the 10 most dominant TCR Vα2 nucleotide sequences.

2. B

   Length of TCR CDR3α2 nucleotide sequences (virtual spectratypes).

3. C

   Relative frequencies of the 10 most dominant TCR Vα2 nucleotide sequences.

4. D

   Lorenz curves of Vα2 TCR repertoires.

5. E, F

   Gini‐TCR skewing and Shannon diversity indexes estimated as described in Materials and Methods section.

Data information: In (A–C and E, F), histograms represent mean values (± s.e.m., n = 4 mice/group) with each symbol referring to an individual mouse. In (D), symbols represent mean values (± s.e.m., n = 4 mice/group).

Figure 6. TRAJ gene segments used by env‐reactive T cells purified from control and Treg‐depleted immune mice

T cells from control and anti‐CD25‐treated immune mice were stimulated as described in the legend to Fig 3 and used for analysis of the Vα2 TCR repertoire.

1. A

   The sum of frequencies of the 10 most dominant TCR Vα2 nucleotide sequences.

2. B

   Virtual spectratypes showing the length of TCR CDR3α2 nucleotide sequences.

3. C–E

   Virtual spectratypes illustrating the relative frequencies of J‐gene usage by the 10 most dominant TCR Vα2 nucleotide sequences from (C) naïve (n = 4 mice), (D) immunized, Treg‐sufficient (n = 27 mice) and (E) immunized, Treg‐depleted TCRβ‐tg mice (n = 27 mice).

4. F

   The relative frequencies of the 10 most dominant TCR Vα2 nucleotide sequences in both groups of immune animals.

5. G

   Lorenz curves of Vα2 TCR repertoire in control and Treg‐depleted mice.

6. H, I

   Gini‐TCR skewing and Shannon diversity indexes.

Data information: In (C), results are from one experiment and in (D, E) from five independent experiments. In (A, B, F, H, I), histograms represent mean values (± s.e.m., n = 27 mice/group, Mann–Whitney test), each symbol referring to an individual mouse. In (G), the symbols represent mean values (± s.e.m., n = 27 mice/group). In (A, B, and F–I), results are from five independent experiments.

Figure EV3. TRAJ gene segments used by naïve and antigen‐activated conventional and regulatory T cells

FoxP3‐expressing (Tregs) and FoxP3‐non‐expressing (Tconv) T cells were purified from naïve Foxp3^DTRTCRβ‐tg mice or from antigen‐stimulated cultures established using CD4‐expressing T cells purified from immunized Foxp3^DTRTCRβ‐tg mice (see protocol described in the legend to Fig 3). Following flow cytometry‐based purification, Tconv and Tregs cells were used for high‐throughput sequencing of CD3Vα2 regions.

1. A, B

   Virtual spectratypes illustrating the relative frequencies of J‐gene usage by the 10 most dominant TCR Vα2 nucleotide sequences from naïve and antigen‐activated FoxP3‐negative (upper panel) and FoxP3‐positive (lower panel) T cells (n = 6 mice for the naïve group and n = 10 mice for the immunized group).

Figure EV4. TCR Vα2 repertoire analysis of naïve and antigen‐activated conventional and regulatory T cells

Conventional (Tconv, left panels) and regulatory (Tregs, right panels) naïve and antigen‐stimulated T cells were purified as described in the legend to Fig EV3. The Vα2 TCR repertoire was analyzed using the criteria described in the legend to Fig 6.

1. The relative frequencies of the 10 most dominant TCR Vα2 nucleotide sequences.

2. Lorenz curves of Vα2 TCR repertoire in Tconv and Tregs.

3. Gini‐TCR skewing and Shannon diversity indexes

4. Virtual spectratypes showing the length of TCR CDR3α2 nucleotide sequences.

Data information: In (A, C, and D), histograms represent mean values (± s.e.m., Mann–Whitney test), each symbol referring to an individual mouse. In (B), the symbols represent mean values (± s.e.m.). In (A–D), n = 6 mice for the naïve group and n = 10 mice for the immunized group.

Figure EV5. Diphtheria toxin‐mediated Treg depletion in FoxP3DTR mice leads to an expanded antigen‐specific TCR repertoire in response to peptide immunization

Env_122–141 peptide‐pulsed splenic dendritic cells (DC) were administrated into the footpads of FoxP3DTR TCRβ‐tg or TCRβ‐tg mice previously treated (DT) or not (PBS) with diphtheria toxin as described in Materials and Methods section. Five days later, purified CD4⁺ T cells from draining lymph nodes were tested ex vivo for Ki67 expression by flow cytometry and further cultured for 48 h with splenic DC and 10⁻⁶ M of env_122–141 peptide. In vitro‐restimulated cells were used for high‐throughput sequencing of CDR3α2 region.

1. A

   Mean percentage of Ki67‐expressing CD4⁺ T cells.

2. B, C

   The sum of frequencies and the relative frequencies of the 10 most dominant TCR Vα2 nucleotide sequences.

3. D

   Lorenz curves of Vα2 TCR repertoires.

4. E, F

   Gini‐TCR skewing and Shannon diversity indexes.

5. G

   Virtual spectratypes illustrating the TCR CDR3α2 length from DT‐treated, immunized TCRβ‐tg (black bars), PBS‐treated, immunized FoxP3DTR TCRβ‐tg (green bars) and DT‐treated, immunized FoxP3DTR TCRβ‐tg mice (magenta bars).

6. H–J

   Virtual spectratypes of the relative frequencies of J‐gene usage by the 10 most dominant TCR Vα2 nucleotide sequences in (H) DT‐treated, immunized TCRβ‐tg (n = 4), (I) PBS‐treated, immunized FoxP3DTR TCRβ‐tg (n = 4) and (J) DT‐treated, immunized FoxP3DTR TCRβ‐tg mice (n = 7).

7. K

   Virtual spectratypes illustrating the length of TCR CDR3α2 nucleotide sequences in naïve, PBS vs. DT‐treated FoxP3DTR TCRβ‐tg mice.

8. L, M

   Virtual spectratypes of the relative frequencies of J‐gene usage by the 10 most dominant TCR Vα2 nucleotide sequences in naïve, (L) PBS (n = 3) vs. (M) DT‐treated (n = 2) FoxP3DTR TCRβ‐tg mice.

Data information: In (A–C, E–G, and K), histograms represent mean values (± s.e.m., n = 2–7 mice/group, Mann–Whitney test) with each symbol depicting an individual mouse. In (D), the symbols represent mean values (± s.e.m., n = 2–7 mice/group). Results are from two independent experiments.

Figure 7. CD86 blockade limits the TCR repertoire of antigen‐reactive cells

Groups of TCRβ‐tg mice were immunized by in vivo transfer of env_122–141 peptide‐pulsed DC following an injection with IgG or anti‐CD86 mAbs. Following in vitro restimulation (according to the protocol described in the legend to Fig 3), cells were analyzed by flow cytometry and used for high‐throughput sequencing of CD3Vα2 regions.

1. A, B

   Representative FACS contour plots and mean percentage of Ki67‐expressing cells in gated Vα2⁺ or Vα2⁻ CD4⁺ T cells.

2. C, D

   The sum of frequencies and the relative frequencies of the 10 most dominant TCR Vα2 nucleotide sequences.

3. E

   Lorenz curves of Vα2 TCR repertoires in naïve and immunized mice in the presence or absence of anti‐CD86 mAbs.

4. F, G

   Gini‐TCR skewing and Shannon diversity indexes.

5. H–J

   Virtual spectratypes illustrating (H) the TCR CDR3α2 length, (I) the relative frequencies of J‐gene usage by the 10 most dominant TCR Vα2 nucleotide sequences in control‐treated, immunized TCRβ‐tg mice (n = 9) and (J) anti‐CD86‐treated, immunized TCRβ‐tg mice (n = 9).

Data information: In (B–D and F–H), histograms represent mean values (± s.e.m., n = 9 mice/group, Mann–Whitney test) with each symbol referring to an individual mouse. In (E), the symbols represent mean values (± s.e.m., n = 9 mice/group). Results are from two independent experiments.