Concomitant deletion of Ptpn6 and Ptpn11 in T cells fails to improve anticancer responses

Abstract

Anticancer T cells acquire a dysfunctional state characterized by poor effector function and expression of inhibitory receptors, such as PD-1. Blockade of PD-1 leads to T cell reinvigoration and is increasingly applied as an effective anticancer treatment. Recent work challenged the commonly held view that the phosphatase PTPN11 (known as SHP-2) is essential for PD-1 signaling in T cells, suggesting functional redundancy with the homologous phosphatase PTPN6 (SHP-1). Therefore, we investigated the effect of concomitant Ptpn6 and Ptpn11 deletion in T cells on their ability to mount antitumour responses. In vivo data show that neither sustained nor acute Ptpn6/11 deletion improves T cell-mediated tumor control. Sustained loss of Ptpn6/11 also impairs the therapeutic effects of anti-PD1 treatment. In vitro results show that Ptpn6/11-deleted CD8⁺ T cells exhibit impaired expansion due to a survival defect and proteomics analyses reveal substantial alterations, including in apoptosis-related pathways. These data indicate that concomitant ablation of Ptpn6/11 in polyclonal T cells fails to improve their anticancer properties, implying that caution shall be taken when considering their inhibition for immunotherapeutic approaches.

Keywords: PD-1 checkpoint blockade; Ptpn11; Ptpn6; T cell exhaustion.

Figure 1. CD4cre Ptpn6 ^fl/fl mice and CD4cre Ptpn6/11 ^fl/fl mice do not exhibit superior antitumor immunity

1. A

   The schematic illustrates CD4cre‐mediated deletion of Ptpn6 or Ptpn6/11 during thymic T cell development; double‐negative thymocytes (DN) and double‐positive thymocytes (DP) are depicted. Created with Biorender (Biorender.com).

2. B

   Total T cells were enriched from spleens of Ptpn6 ^fl/fl /Ptpn11 ^fl/flWbm, CD4cre Ptpn6 ^fl/fl /Ptpn11 ^fl/flWbm, CD4cre Ptpn6 ^fl/fl mice. Expression of Ptpn6 and Ptpn11 was tested by immunoblot in cell lysates, Gapdh was used as loading control.

3. C, D

   CD4cre Ptpn6 ^fl/fl and control mice were subcutaneously injected with MC38 cells. Tumor growth in individual mice is shown for the indicated genotypes and treatments; number of mice eradicating the tumor is shown within the graphs (C). Survival curves are shown (D).

4. E, F

   A representative flow cytometry plot (E), spleen cellularity, CD4⁺ T cell (gated as TCRβ⁺ CD4⁺) numbers, CD8⁺ T cell (gated as TCRβ⁺ CD8⁺) numbers, and percentages of CD44⁺ T cells (F) from the spleens of CD4cre Ptpn6 ^fl/fl /Ptpn11 ^fl/flWbm and control mice are shown.

5. G, H

   Tumor growth in individual mice challenged with MC38 and subsequently treated with anti‐PD‐1 antibody or isotype control is shown for each indicated genotype; number of mice eradicating the tumor is shown within the graphs (G). Survival curves of CD4cre Ptpn6 ^fl/fl /Ptpn11 ^fl/flWbm and control mice is shown (H).

6. I, J

   Ten to twelve days following MC38 tumor inoculation and treatment with anti‐PD‐1 antibody or isotype control, CD4cre Ptpn6/11 ^wt/wt and CD4cre Ptpn6 ^fl/fl /Ptpn11 ^fl/flWbm mice were sacrificed, and TILs analyzed. Graphs depict frequencies of CD8⁺ T cells (gated as CD45⁺ TCRβ⁺ CD8⁺) expressing IFN‐γ and TNFα upon re‐stimulation (I) and percentages of CD8⁺ T cells (J).

Data information: Results depict n = 8–10 mice/group (C, D) and n = 8–11 mice/group (G, H); statistical significance was calculated by log‐rank (Mantel‐Cox) test (D, H). Results depict mean ± SEM of n = 10–12 mice/group (F) and of n = 9–14 mice/group (I, J). Student's t‐test (unpaired, two‐tailed) was used to compare differences between experimental groups (F, I, J). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are available online for this figure.

Figure EV1. Peripheral T cell compartment characterization of CD4cre Ptpn6 ^fl/fl and CD4cre Ptpn6 ^fl/fl /Ptpn11 ^fl/fl mice

1. A, B

   T cells from spleen (A) and inguinal lymph nodes (iLN) (B) of CD4cre Ptpn6 ^fl/fl and control mice were analyzed. Cellularity, CD4⁺ and CD8⁺ T cell numbers (gated as TCRβ⁺ CD4⁺ or CD8^+, respectively), percentages of CD44 high T cells are shown; regulatory T cell (Treg) numbers and frequency (gated as TCRβ⁺ CD4⁺ Foxp3⁺) are illustrated for the spleen.

2. C

   Cellularity from thymus and number of CD4 and CD8 double‐negative, double‐positive, CD4 or CD8 single‐positive thymocytes (gated on lineage⁻; single‐positives, an additional gate on TCRβ⁺ was performed) of CD4cre Ptpn6 ^fl/fl /Ptpn11 ^fl/flWbm and control mice are depicted.

3. D

   Treg number and frequency (gated as TCRβ⁺ CD4⁺ Foxp3⁺) are illustrated for the spleen of CD4cre Ptpn6 ^fl/fl /Ptpn11 ^fl/flWbm and control mice.

4. E

   Cellularity, CD4⁺ and CD8⁺ T cell numbers, as well as percentages of CD44 high T cells from the iLN of CD4cre Ptpn6 ^fl/fl /Ptpn11 ^fl/flWbm and control mice are shown.

5. F

   Survival curves of CD4cre Ptpn6 ^fl/fl /Ptpn11 ^fl/flBgn and control mice challenged with MC38.

6. G–I

   Ten to twelve days following MC38 tumor inoculation, isotype‐treated CD4cre Ptpn6/11 ^wt/wt and CD4cre Ptpn6 ^fl/fl /Ptpn11 ^fl/flWbm mice were sacrificed. Graph depicts the frequencies of PD‐1⁺ CD8⁺ T cells (gated as CD45⁺ TCRβ⁺ CD8⁺) in the tumor (G). Frequencies of CD4⁺ T cells (gated as CD45⁺ TCRβ⁺ CD4⁺) expressing IFN‐γ and TNFα upon re‐stimulation (H) and percentages of CD4⁺ T cells in the tumor are shown (I).

Data information: Results illustrate mean ± SEM of n = 7–8 mice/group (A, B), of n = 10–12 mice/group (C–E), or of n = 9–14 mice/group (G–I). Student's t‐test (unpaired, two‐tailed) was used to compare differences between experimental groups (A–E, G–I). Results depict n = 11 mice/group; statistical significance was calculated by log‐rank (Mantel‐Cox) test (F). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are available online for this figure.

Figure 2. Deletion of Ptpn6/11 in effector cells does not lead to enhanced antitumour response

1. A

   The schematic illustrates GzmBcre‐mediated deletion of Ptpn6/11 following cancer challenge; CD8⁺ T cells and cytotoxic T lymphocytes (CTLs) are depicted. Created with Biorender (Biorender.com).

2. B

   Ptpn6 and Ptpn11 copy number, as estimated through high resolution mass spectrometry in GzmBcre Ptpn6/11 ^fl/fl and control CTLs.

3. C, D

   GzmBcre R26R‐YFP reporter mice were injected subcutaneously with MC38 cells and analyzed after 11 days. Graphs show the percentage of YFP⁺ cells among CD45⁺ CD8⁺ TILs (C) and the percentage of PD‐1⁺ cells among CD45⁺ CD8⁺ YFP⁺ TILs (D). Results depict n = 4 mice (C, D).

4. E, F

   GzmBcre Ptpn6/11 ^fl/fl and control Ptpn6/11 ^fl/fl mice were subcutaneously injected with MC38 cells. Survival curves and statistical comparisons between different groups are shown (E). The tumor start depicts the time (days) following tumor engraftment after which the tumor is palpable, while tumor endpoint depicts the time (days) from tumor start until the reaching the maximal allowed tumor size (F).

Data information: Results depict mean ± SD of n = 3 biological replicates (B), mean ± SEM of n = 4 biological replicates (C, D), mean ± SEM (F) of n = 11–12 mice/group (E, F); statistical significance was calculated by log‐rank (Mantel‐Cox) test (E), and Student's t‐test (unpaired, two‐tailed) was used to compare differences between experimental groups (B, F). **P ≤ 0.01, ***P ≤ 0.001. Source data are available online for this figure.

Figure EV2. Peripheral T cell compartment characterization of GzmBcre Ptpn6/11 ^fl/fl mice

1. A–D

   Spleen and iLN of GzmBcre Ptpn6/11 ^fl/fl mice and control mice were analyzed. Cellularity, CD4⁺ and CD8⁺ T cell numbers are depicted for spleen (A) and iLN (B). Percentages of CD44 high of CD4⁺ and CD8⁺ T cells are shown for spleen (C) and iLN (D). Results depict n = 6–7 mice/group (A, B) and n = 4 mice/group for percentages of CD44 high cells (C, D). Student's t‐test (unpaired, two‐tailed) was used to compare differences between experimental groups.

2. E

   Tumor growth in individual mice challenged with MC38 is shown for the indicated genotypes; number of mice eradicating the tumor is shown within the graphs.

Source data are available online for this figure.

Figure EV3. Characterization of CTLs from Ptpn6/11 ^fl/fl and GzmBcre Ptpn6/11 ^fl/fl mice

CD8⁺ cytotoxic lymphocytes were generated from splenocytes of the indicated mice by anti‐CD3, anti‐CD28, and IL‐12 stimulation and maintained in IL‐2.

1. A, B

   CD8⁺ T cells were analyzed by flow cytometry for surface expression of GITR and CD71 after 2 days (A) and for surface expression of GITR, CD71, CD69, and CD44 after 5 days (B); a quantification of these parameters is shown in the graphs (A and B).

2. C

   Graphs depict the percentages of cells in G1, S, and G2 phase as measured by flow cytometry‐based cell cycle analysis of Ptpn6/11 ^fl/fl and GzmBcre Ptpn6/11 ^fl/fl CTLs (gated on CD8⁺ T cells) at the indicated days.

Data information: Results depict mean ± SD of n = 3 biological replicates and Student's t‐test (unpaired, two‐tailed) was used to compare differences between experimental groups (A–C). *P ≤ 0.05. Source data are available online for this figure.

Figure 3. Deletion of Ptpn6 and Ptpn11 impacts on CD8⁺ T cell proteome and cell survival

CTLs were generated from splenocytes of GzmBcre Ptpn6/11 ^fl/fl and Ptpn6/11 ^fl/fl mice by anti‐CD3, anti‐CD28, and IL‐12 stimulation and maintained in IL‐2.

1. A, B

   CD8⁺ T cells (gated DAPI⁻ cells) were analyzed by flow cytometry for size, granularity, and surface expression of CD25 and PD‐1 after 2 days (A) and after 5 days (B); a quantification of these parameters is shown in the graphs (A, B) and a representative flow cytometry plot is shown for FSC and SSC (A).

2. C

   Numbers of CTLs (gated on CD8⁺ T cells) from Ptpn6/11 ^fl/fl or GzmBcre Ptpn6/11 ^fl/fl mice were counted daily by flow cytometry with the addition of DAPI to monitor dead cells.

3. D

   Quantitative high‐resolution mass spectrometry was used to resolve the proteome of Ptpn6/11 ^fl/fl and GzmBcre Ptpn6/11 ^fl/fl CTLs (day 7). The plot depicts protein copy number in control and GzmBcre Ptpn6/11 ^fl/fl cells. Significantly altered proteins are indicated in red; selected examples are annotated. Results are based on n = 3 biological replicates and two‐tailed, unequal‐variance t‐test on log10 transformed copy number per cell values was used to compare differences between experimental groups.

4. E

   GO Term analysis shows the enriched pathways with at least 23 proteins and a P‐value < 0.05, % indicate the % altered genes on the total genes in a given pathway.

5. F

   Graph shows the percentage of sub‐G1 cells as measured by flow cytometry‐based cell cycle analysis of Ptpn6/11 ^fl/fl and GzmBcre Ptpn6/11 ^fl/fl CTLs (gated on CD8⁺ T cells) at the indicated days.

6. G

   Graph illustrates the percentage of DAPI⁺ dead cells of Ptpn6/11 ^fl/fl and GzmBcre Ptpn6/11 ^fl/fl CTLs (gated on CD8⁺ T cells) at the indicated days as measured by flow cytometry.

7. H

   Protein copy number of Tnfrsf1b and Mcl‐1 in GzmBcre Ptpn6/11 ^fl/fl and control CTLs, as estimated through high resolution mass spectrometry.

Data information: Results depict mean ± SD of n = 3 (A, B, F, H) or 4 (C, G) biological replicates and Student's t‐test (unpaired, two‐tailed) was used to compare differences between experimental groups (A–C, F–H). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are available online for this figure.