Adoptive T cell therapy cures mice from active hemophagocytic lymphohistiocytosis (HLH)

Abstract

Primary hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome caused by impaired lymphocyte cytotoxicity. First-line therapeutic regimens directed against activated immune cells or secreted cytokines show limited efficacy since they do not target the underlying immunological problem: defective lymphocyte cytotoxicity causing prolonged immune stimulation. A potential rescue strategy would be the adoptive transfer of ex vivo gene-corrected autologous T cells. However, transfusion of cytotoxicity-competent T cells under conditions of hyperinflammation may cause more harm than benefit. As a proof-of-concept for adoptive T cell therapy (ATCT) under hyperinflammatory conditions, we transferred syngeneic, cytotoxicity-competent T cells into mice with virally triggered active primary HLH. ATCT with functional syngeneic trigger-specific T cells cured Jinx mice from active HLH without life-threatening side effects and protected Perforin-deficient mice from lethal HLH progression by reconstituting cytotoxicity. Cured mice were protected long-term from HLH relapses. A threshold frequency of transferred T cells with functional differentiation was identified as a predictive biomarker for long-term survival. This study is the first proof-of-concept for ATCT in active HLH.

Keywords: adoptive T cell therapy; hemophagocytic lymphohistiocytosis; hyperinflammation; virus-specific T cells.

Figure 1. Nonfatal active HLH in Jinx mice on day 15 post infection

Jinx mice and heterozygous littermates (WT) were infected with 200 pfu LCMV‐WE intravenously (i.v.).

1. A, B

   Bodyweight (A) and survival (B) of mice were monitored for 5 weeks (n (A) = 43 Jinx, 52 WT; n (B) = 88 Jinx, 73 WT).

2. C

   Spleen and liver sections (day 15–21 post infection (p.i.)) were stained with hematoxylin and eosin (HE) (n = 6–7 mice per group).

3. D, E

   Spleen and liver weight in relation to bodyweight were analyzed on day 15 p.i. (n = 17 Jinx, 9 WT).

4. F, G

   Degranulation capacity of CD8 T cells on day 8 p.i. after restimulation with LCMV‐GP_33–41: Frequency of CD107a⁺ of all IFNγ⁺ CD8 T cells. The following analyses were performed on day 15 p.i. (n = 12 Jinx, 14 WT).

5. H–K

   Ear temperature, frequency of white blood cells (WBC), concentration of hemoglobin (HGB), and frequency of platelets (PLT) in blood (n = 15–17 Jinx, 7–8 WT).

6. L–Q

   Serum concentration of ferritin, soluble CD25, triglycerides, glutamate‐pyruvate transaminase (GPT), lactate dehydrogenase (LDH) and interferon γ (IFNγ) (n = 15–17 Jinx, 6–9 WT).

7. R

   Virus titres of spleen, liver, lung, brain, and kidney (n = 15 Jinx, 9 WT).

Data information: Horizontal lines in graphs represent mean values. Horizontal dashed line (Q, R) indicates the detection limit. Scale bars are 200 μm long. Data are mean ± SEM with n = 6–17 per group in 2–4 independent experiments. Statistics: unpaired t‐test (D, E, H, K, N, O), log‐rank test (B), Mann–Whitney test (B, F, I, J, L, M, P, Q), ns (not significant) P > 0.05; ****P ≤ 0.0001.

Figure 2. Adoptive T cell therapy cures Jinx mice from active HLH

Jinx mice and heterozygous littermates (WT) were infected with 200 pfu LCMV‐WE i.v. On day 15 p.i., 4 × 10⁶ purified CD3 T cells from LCMV‐immune WT mice were transferred to Jinx mice (Jinx + ATCT).

1. A

   Bodyweight was monitored for 5 weeks p.i. (n = 42–52 per group).

2. B, C

   Analyses performed on day 35 p.i.: (B) Liver sections stained with hematoxylin and eosin (HE) (n = 3–5 in 1 experiment) and (C) ear temperature (n = 12 Jinx, 10 Jinx + ATCT, 15 WT).

3. D–F

   Frequency of white blood cells (WBC), concentration of hemoglobin (HGB), and frequency of platelets (PLT) in blood (n = 17 Jinx, 16 Jinx + ATCT, 21 WT).

4. G–L

   Serum concentration of ferritin, soluble CD25, triglycerides, GPT, LDH, and IFNγ (n = 8–15 Jinx, 10–13 Jinx + ATCT, 9–15 WT).

5. M, N

   Spleen and liver weight in relation to bodyweight (n = 17 Jinx, 16 Jinx + ATCT, 22 WT).

6. O

   Virus titres in liver, lung, brain, and kidney (n = 19 Jinx, 16 Jinx + ATCT, 24 WT).

Data information: Horizontal lines in graphs represent mean values. Horizontal dashed line (L, O) indicates the detection limit. Scale bars are 200 μm long. Data are mean ± SEM with n = 8–24 per group in at least three independent experiments. Statistics: unpaired t‐test (A, G, H, J), Mann–Whitney test (C, D, E, F, I, K, L, M, N), ns P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Figure EV1. CD8 T cell phenotype in HLH mouse models and FHL patients

Left column: CD8 T cells obtained from 1°HLH patients (1°HLH) were analyzed by flow cytometry in comparison to healthy donors (HD). Right column: Jinx and PKO mice (1°HLH) were infected with 200 pfu LCMV‐WE intravenously and analyzed by flow cytometry on day 12–15 p.i. in comparison to noninfected wild‐type mice (WT).

1. A, B

   The frequency of CD8 T cells double negative for KLRG1 and CD127 (KLRG1⁻CD127⁻) was determined (A) in blood of 1°HLH patients, HD and (B) in the spleen of 1°HLH mouse models, as well as noninfected WT mice (n = 17 1°HLH patients, n = 11 HD, n = 30 1°HLH mice, n = 13 WT mice).

2. C, D

   Frequency of CD8 T cells expressing PD‐1 was determined (C) in blood of 1°HLH patients in comparison to HD (D) and in the spleen of 1°HLH mouse models, as well as noninfected WT mice (n = 17 1°HLH patients, n = 11 HD, n = 21 1°HLH mice, n = 11 WT mice).

3. E

   Expression of CD45RA and CCR7 on CD8 T cells was determined in blood of 1°HLH patients and HD and four populations were distinguished: CD45RA⁺CCR7⁺ termed “naïve”, CD45RA⁻CCR7⁺ termed “T_CM”, CD45RA⁻CCR7⁻ termed “T_EM” and CD45RA⁺CCR7⁻ (n = 17 1°HLH patients, n = 11 HD).

4. F

   Expression of CD62L and CD44 on CD8 T cells was determined in the spleen of 1°HLH mouse models, and noninfected WT mice and four populations were distinguished: CD44⁻CD62L⁺ termed “naïve”, CD44⁺CD62L⁺ termed “T_CM”, CD44⁺CD62L⁻ termed “T_EM” and CD44⁻CD62L⁻ (n = 19 1°HLH mice, n = 9 WT mice).

Data information: Horizontal lines in graphs represent mean values. Data are mean ± SEM. Statistics: unpaired t‐test (B, D), Mann–Whitney test (A, C). ****P ≤ 0.0001. Source data are available online for this figure.

Figure 3. T cell differentiation after ATCT in active HLH

Jinx mice and heterozygous littermates (WT) were infected with 200 pfu LCMV‐WE i.v. On day 15 p.i., 4 × 10⁶ purified CD3 T cells from LCMV‐immune WT mice were transferred to Jinx mice (Jinx + ATCT). Transferred CD8 T cells (trsf.) were distinguished from endogenous CD8 T cells (endog.). As controls, Jinx and WT mice were left untreated.

1. A–C

   On day 35 p.i., endogenous and transferred CD8 T cells (columns I and II), as well as LCMV‐GP_33–41‐specific CD8 T cells (column III) in the spleen, were analyzed by flow cytometry: (A) frequency of KLRG1⁺ and/or CD127⁺, (B) PD‐1⁺/LAG3⁺ and (C) TCF‐1⁻/TIM3⁺ CD8 T cells (C) The same analyses were performed more than 100 days after therapy/115 days after infection (column IV).

Data information: FACS plots are representative of the respective mouse groups. Horizontal lines in graphs represent mean values. Data are mean ± SEM with n = 8–15 per group in 2–5 independent experiments. Detailed information n: A. (II) n = 12 Jinx, 15 Jinx + ATCT (15× trsf cells), 15 WT in 5 experiments; (III) n = 12 Jinx, 13 Jinx + ATCT (13× trsf cells), 15 WT in 5 experiments; (IV) n = 15 Jinx + ATCT (15× trsf cells), 8 WT in 3 experiments. B. (II) n = 14 Jinx, 15 Jinx + ATCT (13× trsf cells), 18 WT in 5 experiments; (III) n = 14 Jinx, 15 Jinx + ATCT (13× trsf cells), 18 WT in 3 experiments; (IV) n = 15 Jinx + ATCT (15× trsf cells), 8 WT in 3 experiments. C. (II) n = 9 Jinx, 11 Jinx + ATCT (9× trsf cells), 13 WT in 4 experiments; (III) n = 9 Jinx, 11 Jinx + ATCT (9× trsf cells), 13 WT in 4 experiments; (IV) n = 9 Jinx + ATCT (9× trsf cells), 5 WT in 2 experiments. Statistics: unpaired t‐test (A column IV, B column II, C column II and III), Mann–Whitney test (A column II, III, B column III and IV, C column IV), ns P > 0.05; *P ≤ 0.05; ****P ≤ 0.0001.

Figure EV2. Adoptive T cell therapy with naïve or LCMV‐effector T cells in active HLH is not successful

1. Purified CD3 T cells used for ATCT were analyzed regarding the frequency of CD4 and CD8 T cells and the frequency of LCMV‐GP_33–41‐specific CD8 T cells among CD8 T cells. Individual dots represent separate transfers/experiments (n = 7).

2. Jinx mice (Jinx) and heterozygous littermates (WT) were infected with 200 pfu LCMV‐WE intravenously. On day 5–15 p.i., mice remained untreated (Jinx, n = 8; WT, n = 10) or 1 × 10⁷ purified CD3 T cells/lymphocytes were transferred to Jinx mice (Jinx + eff, n = 9). Alternatively, Jinx mice received on day 15 p.i. a transfer with 1 × 10⁷ CD3 T cells from uninfected wild‐type mice (Jinx + naïve, n = 4).

Data information: Horizontal lines in graphs represent mean values. Data are mean ± SEM with n = 4–10 mice in 1–3 experiments.

Figure 4. Adoptive T cell therapy cures Jinx mice from HLH and protects against HLH relapses

Jinx mice and heterozygous littermates (WT) were infected with 200 pfu LCMV‐WE i.v. On day 15 p.i. 4 × 10⁶ CD3 T cells from LCMV‐immune WT mice were transferred to Jinx mice (Jinx + ATCT). As controls, Jinx and WT mice were left untreated. A further experimental group was re‐challenged with 10⁵–10⁶ pfu LCMV‐Armstrong intraperitoneally (Jinx + ATCT+challenge) more than 45 days after therapy.

1. A

   Mouse survival was followed for 22 weeks p.i. or postchallenge (n = 88 Jinx, 43 Jinx + ATCT, 7 Jinx + ATCT + challenge; n (survival until week 22 p.i./challenge) = 2/38 Jinx, 21/22 Jinx + ATCT, 7/7 Jinx + ATCT + challenge in ≥ 2 experiments).

2. B, C

   Bodyweight and virus titres in week 17 p.i. or postchallenge (n = 12 Jinx, 21 Jinx + ATCT, 7 Jinx + ATCT + challenge, 17 WT).

3. D

   Frequency of transferred CD8 T cells in the spleens of recipients 1 day (n = 4), 3 weeks (n = 16) and 15 weeks (n = 21) after therapy or 15 weeks after challenge (n = 7).

Data information: Horizontal lines in graphs represent mean values. Horizontal dashed line (C) indicates the detection limit. Data are mean ± SEM with n = 7–21 in ≥ 2 experiments. Statistics: log‐rank test (A), Mann–Whitney test (B, D), ***P ≤ 0.001; ****P ≤ 0.0001. Source data are available online for this figure.

Figure EV3. Expression of transcription factors and functionality of T cells after adoptive T cell therapy in active HLH

Jinx mice (Jinx) and heterozygous littermates (WT) were infected with 200 pfu LCMV‐WE intravenously. On day 15 post infection (p.i.), mice remained untreated (Jinx, WT) or 4 × 10⁶ purified CD3 T cells from LCMV‐immune WT mice were transferred to Jinx mice (Jinx + ATCT). Transferred CD8 T cells (trsf.) were distinguished from endogenous CD8 T cells (endog.).

1. A, B

   On day 20 after therapy, endogenous and transferred CD8 T cells (column II), as well as LCMV‐GP_33–41‐specific CD8 T cells (column III), were analyzed by flow cytometry: frequency of TCF‐1⁺ or TOX⁺. The same analyses were performed more than 100 days after therapy (column IV).

2. C

   Splenocytes were restimulated with LCMV‐GP_33–41. The frequencies of transferred WT CD8 T cells in Jinx recipients and WT CD8 T cells expressing IFNγ and TNFα or IFNγ and CD107a after restimulation were determined on day 20 or > 100 days after therapy start (C, columns II, IV).

Data information: Horizontal lines in graphs represent mean values. ns P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Data are mean ± SEM with n (A–C) 3–19 mice in 1–5 experiments. Statistics: unpaired t‐test (A column II, III, IV; B column IV), Mann–Whitney test (B column II, III; C column II, IV). Detailed information n: A. (II) n = 9 Jinx, 11 Jinx + ATCT (9× trsf. cells), 11 WT in 4 experiments; (III) n = 4 Jinx, 5 Jinx + ATCT (3× trsf. cells), 6 WT in 2 experiments; (IV) n = 10 Jinx + ATCT, 3 WT in 2 experiments. B. (II) n = 9 Jinx, 11 Jinx + ATCT (6× trsf. cells), 11 WT in 4 experiments; (III) n = 5 Jinx, 6 Jinx + ATCT (3× trsf. cells), 7 WT in 2 experiments; (IV) n = 9 Jinx + ATCT (6× trsf. cells), 3 WT in 2 experiments. C. (II) IFNγ/TNFα: n = 16 trsf. cells in Jinx + ATCT, 16 WT in 5 experiments; (II) IFNγ/CD107a: n = 11 trsf. cells in Jinx + ATCT, 10 WT in 3 experiments; (II) IFNγ/TNFα: n = 6 trsf. cells in Jinx + ATCT, 3 WT in 1 experiment; (IV) IFNγ/CD107a: n = 6 trsf. cells in Jinx + ATCT, 3 WT in 1 experiment.

Figure 5. Successful ATCT does not strictly correlate with high bodyweight

Jinx mice were infected with 200 pfu LCMV‐WE i.v.

1. A, B

   On day 15 p.i., mice remained untreated or received 1 × 10⁷ total lymphocytes (Jinx + lym), 4 × 10⁶ purified CD3 T cells (Jinx + CD3), or 4 × 10⁶ purified CD8 T cells (Jinx + CD8) from LCMV‐immune wild‐type mice. (A) Virus titres (n = 19 Jinx, 5 Jinx + lymph, 16 Jinx + CD3, 8 Jinx + CD8) and (B) bodyweight on day 20 after therapy (n = 43 Jinx, 19 Jinx + lym, 42 Jinx + CD3, 11 Jinx + CD8).

2. C, D

   On day 15 p.i., Jinx mice remained untreated or received a transfer of 4 × 10⁶, 1 × 10⁶, or 1 × 10⁵ purified CD3 T cells from LCMV‐immune wild‐type mice. (C) Virus titres and (D) bodyweight on day 20 after therapy (n = 7 Jinx, 8 Jinx + 40 × 10⁵, 9 Jinx + 10 × 10⁵, 8 Jinx + 1 × 10⁵).

Data information: Horizontal lines in graphs represent mean values. Horizontal dashed line (A, B) indicates the detection limit. Data are mean ± SEM with n = 5–19 per group in 1–8 independent experiments (A–D). Statistics: Mann–Whitney test (B, D), ns P > 0.05; **P ≤ 0.01; ****P ≤ 0.0001.

Figure 6. Frequency of adoptively transferred functional CD8 T cells in Jinx mice predicts therapy success

1. A–D

   Jinx mice were infected with 200 pfu LCMV‐WE i.v. Pooled data from ATCT experiments irrespective of the number and composition of the transferred cell population analyzed ≥ day 20 after therapy. (A) Frequency of “functional” transferred CD8 T cells (funct. trsf. CD8; KLRG1⁺ and/or CD127⁺) of all lymphocytes in the spleen was correlated with virus clearance to determine therapy success. Cured (LCMV‐free) recipients (gray bars) versus not cured (persistently infected) recipients (black bars; n = 99). (B) Procedure described in (A) was repeated for “functional” transferred CD8 T cells, which are PD‐1^lowLAG3^low (n = 92). (C, D) Jinx mice received on day 15 p.i. 1 × 10⁷ lymphocytes (Jinx + lym), 4 × 10⁶ purified CD3 (Jinx + CD3), or 4 × 10⁶ purified CD8 T cells (Jinx + CD8) from LCMV‐immune wild‐type mice. Alternatively, Jinx mice received effector T cells from acutely LCMV‐infected WT mice (day 5–15 p.i.; Jinx + eff). Frequency of transferred “functional” CD8 T cells (PD‐1^lowLAG3^low or CD127⁺ and/or KLRG1⁺) in recipient Jinx mice at the indicated time points after therapy in (C) spleen and (D) blood. Successful LCMV clearance from all organs (cured, open symbol) versus no LCMV clearance (not cured, filled symbol). (n (C) = 10 Jinx + eff. – day 20, 13 Jinx + CD3 – day 20, 15 Jinx + CD3 > day 100, 7 Jinx + lym. > day 100, 4 Jinx + CD8 > day 100). (n (D) = 5 Jinx + eff. – day 20–35, 7 Jinx + lym. – day 20–35, 5 Jinx + CD3 – day 20–35, 12 Jinx + CD3 > day 100, 3 Jinx + CD8 > day 100).

Data information: Horizontal lines in graphs represent mean values. Dotted lines (A–D) indicate thresholds. Data are mean ± SEM with n = 92–99 in 18 independent experiments (A, B) and n = 5–19 in 1–8 independent experiments (C, D). Statistics: Mann–Whitney test (C, D), ns P > 0.05.

Figure 7. Frequency of adoptively transferred functional CD8 T cells in Jinx mice predicts therapy success 10 days post‐therapy

Jinx mice were infected with 200 pfu LCMV‐WE i.v. On day 15 p.i., Jinx mice received a transfer of 4 × 10⁶ or 1 × 10⁵ purified CD3 T cells from LCMV‐immune wild‐type mice.

1. Frequency of “functional” transferred CD8 T cells (funct. trsf. CD8), (KLRG1⁺ and/or CD127⁺ of all lymphocytes) in recipients 10 days after therapy (n = 7 mice per group).

2. Procedure of (A) was repeated for transferred CD8 T cells with low expression of PD‐1 and LAG3 (n = 7 mice per group).

3. Virus titers were determined 10 days after therapy (n = 7 mice per group).

Data information: Dotted lines (A, B) indicate thresholds. Horizontal lines in graphs represent mean values. Data are mean ± SEM with n = 7 mice per group in two experiments. Statistics: unpaired t‐test (A, B), ** P ≤ 0.01; **** P ≤ 0.0001.

Figure 8. Successful adoptive T cell therapy in Perforin‐deficient mice

Perforin‐deficient mice (PKO) and wild‐type controls (WT) were infected with 200 pfu LCMV‐WE i.v. On day 5 p.i. mice remained untreated (PKO, WT) or received 1 × 10⁷ lymphocytes or 4 × 10⁶ purified CD3 T cells from LCMV‐immune wild‐type mice (PKO + ATCT; pooled data). Untreated PKO mice were analyzed on day 12 p.i., PKO mice with transferred cells, and WT mice on day 25–30 after therapy.

1. A

   Bodyweight of PKO, PKO + ATCT and WT mice for 5 weeks after infection (n = 9 PKO, 9 PKO + ATCT, 7 WT).

2. B

   Cell frequency of “functional” (PD‐1^lowLAG3^low) transferred CD8 T cells in the spleen and blood of PKO + ATCT that eliminated LCMV (cured) compared with PKO recipients with insufficient therapy (transfer of 1 × 10⁵ purified CD3 T cells transferred on day 7 p.i., analysis on day 12–19 p.i.) that did not eliminate LCMV (not cured). (n (spleen) = 9 PKO + 4 × 10⁶, n = 6 PKO + 1 × 10⁵), (n (blood) = 5 PKO + 4 × 10⁶, n = 6 PKO + 1 × 10⁵).

3. C

   Virus titres in liver, lung, brain, and kidney (n = 9 PKO, 9 PKO + ATCT, 7 WT).

4. D

   Ear temperature (n = 9 PKO, 9 PKO + ATCT, 7 WT).

5. E–G

   Frequency of white blood cells (WBC), concentration of hemoglobin (HGB) and frequency of platelets (PLT) in blood (n = 9 PKO, 9 PKO + ATCT, 7 WT).

6. H–M

   Serum concentration of ferritin, soluble CD25, triglycerides, glutamate‐pyruvate transaminase (GPT), lactate dehydrogenase (LDH) and interferon γ (IFNγ) (n = 7–9 PKO, 6–9 PKO + ATCT, 6–7 WT).

7. N, O

   Spleen and liver weight were analyzed in relation to bodyweight of mice (n = 8–9 PKO, 9 PKO + ATCT, 7 WT).

Data information: Dotted lines (B) indicate thresholds. Horizontal lines in graphs represent mean values. Data are mean ± SEM with n = 5–10 per group in 2–3 independent experiments. Statistics: unpaired t‐test (B–G, I–L, N), Mann–Whitney test (H, J, M, O), ns P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Figure EV4. Frequency of adoptively transferred functional CD8 T cells in Jinx mice indicates therapy success 20 days post‐therapy

Jinx mice were infected with 200 pfu LCMV‐WE intravenously.

1. A, B

   On day 15 p.i., different numbers of lymphocytes, CD3, CD4, or CD8 T cells were transferred. Analyses ≥ day 20 after therapy. (A) Frequency of “functional” transferred CD8 T cells (funct. trsf. CD8; KLRG1⁺ and/or CD127⁺) of all lymphocytes in recipients that cleared LCMV (cured) or not (not cured; n = 62 “cured”, n = 37 “not cured” in 18 experiments). (B) Procedure of (A) was repeated for “functional” transferred CD8 T cells with low expression of PD‐1 and LAG3. (C, D) On day 15 p.i., Jinx mice remained untreated or received a transfer of 4 × 10⁶, 1 × 10⁶, or 1 × 10⁵ purified CD3 T cells from LCMV‐immune wild‐type mice (n = 60 “cured”, n = 32 “not cured” in 18 experiments).

2. C

   Frequency of “functional” transferred CD8 T cells (funct. trsf. CD8), (KLRG1⁺ and/or CD127⁺ of all lymphocytes) in recipients that cleared LCMV (cured) or not (not cured) 20 days after therapy (n = 8–9 mice per group).

3. D

   Procedure of (C) was repeated for transferred CD8 T cells with low expression of PD‐1 and LAG3 (n = 5–7 mice per group).

Data information: Dotted lines (A–D) indicate thresholds. Horizontal lines in graphs represent mean values. Data are mean ± SEM with n (A, B) 32–62 mice in 18 experiments, n (C, D) 5–9 mice in 3 experiments. Statistics: Mann–Whitney test (A–D). ns P > 0.05; *P ≤ 0.05; ****P ≤ 0.0001. Source data are available online for this figure.

Figure EV5. Readjusted T cell differentiation after adoptive T cell therapy in PKO mice

Perforin‐deficient mice (PKO) and wild‐type controls (WT) were infected with 200 pfu LCMV‐WE intravenously. On day 5 post infection (p.i.), mice remained untreated (PKO, WT) or received an adoptive transfer of 1 × 10⁷ lymphocytes or 4 × 10⁶ purified CD3 T cells from LCMV‐immune wild‐type mice (PKO + ATCT). Transferred CD8 T cells (trsf.) were distinguished from endogenous CD8 T cells (endog.). Untreated PKO mice were analyzed on day 12 p.i., PKO mice with transferred cells, and WT mice on day 25–30 after therapy.

1. A–D

   Endogenous and transferred CD8 T cells were analyzed by flow cytometry: KLRG1⁺ and/or CD127⁺ (A), PD‐1⁺LAG3⁺ (B), TIM3⁺TCF‐1⁻ (C) and TOX⁺ (D) (n = 9 PKO, 9 PKO + ATCT, 7 WT). Data information: Data are mean ± SEM with n (A–D) 7–9 mice in 3 experiments. Statistics: Mann–Whitney test (A–D). ****P ≤ 0.0001.

2. E

   Exemplary gating strategy: (1) gating on lymphocytes; (2) exclusion of doublets; (3) determination of the frequency of CD4 and CD8 T cells, gating on CD8 T cells; (4) discrimination of CD45.1⁺ and CD45.2⁺ CD8 T cells; (5) analysis of CD45.1⁺ or CD45.2⁺ LCMV‐GP_33–41‐specific CD8 T cells.