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. 2024 Feb 1;134(3):e164325.
doi: 10.1172/JCI164325.

PD-1H/VISTA mediates immune evasion in acute myeloid leukemia

Tae Kon Kim  1   2   3   4   5   6 , Xue Han  7   8   9 , Qianni Hu  1 , Esten N Vandsemb  10 , Carly M Fielder  1 , Junshik Hong  11 , Kwang Woon Kim  1 , Emily F Mason  3 , R Skipper Plowman  3 , Jun Wang  12 , Qi Wang  13 , Jian-Ping Zhang  7 , Ti Badri  7 , Miguel F Sanmamed  14 , Linghua Zheng  7   8 , Tianxiang Zhang  7 , Jude Alawa  7 , Sang Won Lee  7 , Amer M Zeidan  6 , Stephanie Halene  6 , Manoj M Pillai  6 , Namrata S Chandhok  15 , Jun Lu  16 , Mina L Xu  17 , Steven D Gore  6   18 , Lieping Chen  5   7
Affiliations

PD-1H/VISTA mediates immune evasion in acute myeloid leukemia

Tae Kon Kim et al. J Clin Invest. .

Abstract

Acute myeloid leukemia (AML) presents a pressing medical need in that it is largely resistant to standard chemotherapy as well as modern therapeutics, such as targeted therapy and immunotherapy, including anti-programmed cell death protein (anti-PD) therapy. We demonstrate that programmed death-1 homolog (PD-1H), an immune coinhibitory molecule, is highly expressed in blasts from the bone marrow of AML patients, while normal myeloid cell subsets and T cells express PD-1H. In studies employing syngeneic and humanized AML mouse models, overexpression of PD-1H promoted the growth of AML cells, mainly by evading T cell-mediated immune responses. Importantly, ablation of AML cell-surface PD-1H by antibody blockade or genetic knockout significantly inhibited AML progression by promoting T cell activity. In addition, the genetic deletion of PD-1H from host normal myeloid cells inhibited AML progression, and the combination of PD-1H blockade with anti-PD therapy conferred a synergistic antileukemia effect. Our findings provide the basis for PD-1H as a potential therapeutic target for treating human AML.

Keywords: Cancer immunotherapy; Costimulation; Immunology; Leukemias; Oncology.

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Figures

Figure 1
Figure 1. PD-1H protein is highly expressed on AML blasts.
(A) Immunohistochemical staining of human PD-1H and PD-L1 in AML. Validation of PD-1H and PD-L1 staining in human placenta (left panels). IHC staining of PD-1H and PD-L1 in human AML BM core biopsies (right panels) (representative photographs, monocytic AML). Original magnification, ×400. Scale bars: 20 mm. (B) Pathologic score of PD-1H and PD-L1 expression in AML BM core biopsies. Scores of 0, 1, 2, and 3 indicate that less than 5%, 5%–20%, 20%–40%, and more than 40% of AML blasts, respectively, showed PD-1H or PD-L1 expression. (C) Flow cytometric analysis of healthy donor (HD) CD34+ cells (far left), AML blasts (either CD34+ or CD33+) (second panel), HD CD11b+ myeloid cells (third panel), and HD CD3+ T cells (far right). (D) Change in (Δ) MFI (MFI in PD-1H staining–MFI in isotype staining). Mean value of ΔMFI in HD CD34+ progenitors versus mean value of ΔMFI in AML CD34+ blasts = 76 ± 26.8 (n = 5) versus 11,469 ± 4,873 (n = 26), P = 0.02. P value determined by Student’s t test. Error bars represent SEM. (E) Flow cytometric analysis of AML subsets (t[8;21], complex karyotype, nonmonocytic, and monocytic). (F) Mean value of ΔMFI in t(8;21) versus in monosomic complex karyotype AML (551 ± 145 [n = 4] versus 9,469 ± 3,880 [n = 8]). P value determined by 1-way ANOVA. Error bars represent SEM. *P < 0.05. (G) Mean value of ΔMFI in nonmonocytic versus monocytic AML (822 ± 155 [n = 19] versus 23,881 ± 9,533 [n = 7]). P value determined by 1-way ANOVA. Error bars represent SEM. *P <0.05.
Figure 2
Figure 2. AML surface PD-1H inhibits T cell infiltration, leading to immune evasion.
(A) Syngeneic mouse leukemia model using tail-vein injection with myeloid leukemia cells (C1498). Mouse leukemia cells expressing PD-1H (C1498FF–PD-1H) or cells not expressing PD-1H (C1498FF-mock) were transplanted into B6 mice and assessed for in vivo leukemia proliferation using bioluminescence. (B) In vivo proliferation of C1498FF-mock versus C1498FF–PD-1H cells in B6 WT mice (n = 7). Radiance indicates the mean value per group and error bars represent SEM. P value determined by Student’s t test at each time point. *P <0.05; ***P < 0.001. These experiments were repeated 3 times. Repeated measures were determined by ANOVA with 2 factors (P > 0.05, no difference among experiments). (C) In vivo proliferation of C1498FF-mock versus C1498FF–PD-1H cells in NSG mice (n = 3) (representative images on day 21 on the right side). Radiance indicates the mean value per group, and error bars represent SEM. P value determined by Student’s t test at each time point. Repeated measures were determined by ANOVA with 2 factors (P > 0.05, no difference among experiments). (D) In vitro growth of C1498FF–PD-1H tumors compared with C1498FF-mock tumors. Statistical analysis was done using Student’s t test. (E) Syngeneic mouse model using s.c. injection with C1498 cells. C1498FF–PD-1H cells or C1498FF-mock cells were s.c. injected into the flanks of B6 mice and the tumor volume was assessed. Mean tumor volume ± SEM. P value determined by Student’s t test at each time point. n = 5 per group; P = 0.07. Mice were sacrificed on day 12, and tumor tissues were removed for mass cytometry assay. (F) Quantification of immune subsets in mass cytometry data in C1498FF–PD-1H tumors compared with C1498FF-mock tumors. n = 5 per group, P value determined by Student’s t test. Error bars represent SEM. *P < 0.05; **P < 0.01.
Figure 3
Figure 3. Host-derived PD-1H induces immune evasion in AML.
(A) Syngeneic mouse leukemia model using tail-vein injection with myeloid leukemia cells (C1498). Mouse leukemia cells (C1498FF-mock) were transplanted into B6 PD-1H WT or PD-1H–KO mice or lineage-specific PD-1H–KO mice. In vivo proliferation was assessed by bioluminescence. (B) In vivo antileukemia effect of genetic deletion of PD-1H in host mice. Radiance indicates the mean value per group, and error bars represent SEM. P value determined by Student’s t test at each time point. n = 5 per group; *P < 0.05. These experiments were repeated 3 times. Repeated measures were determined by ANOVA with 2 factors (P > 0.05, no difference among experiments). (C) In vivo antileukemia effect of myeloid lineage–specific deletion of PD-1H in host mice. Bioluminescence was compared in LysM-Cre+PD-1H-floxed mice with control–PD-1H-floxed mice. Radiance indicates the mean value per group, and error bars represent SEM. P value determined by Student’s t test at each time point. *P < 0.05. n = 9 per group. Representative data from 2 independent experiments were combined. Repeated measures were determined by ANOVA (P > 0.05, no difference among experiments). (D) In vivo antileukemia effect of T cell lineage–specific deletion of PD-1H in host mice. Bioluminescence was compared in Lck-Cre+PD-1H-floxed mice versus control–PD-1H-floxed mice. Radiance indicates the mean value per group, and error bars represent SEM. P value determined by Student’s t test at each time point. Error bars represent SEM. n = 6 per group. Repeated measures were determined by ANOVA (P > 0.05, no difference among experiments).
Figure 4
Figure 4. Anti-mouse PD-1H mAb reverses immune evasion induced by mouse AML surface PD-1H.
(A) PD-1H suppressed T cell activation. Inhibition of OT-1 T cells by mouse PD-1H on 293-KbOVA cells. T cell proliferation was assessed by CFSE dilution. The diluted population was assessed by the percentage of total T cells. (B and C) B6 PD-1H–KO mice were transplanted with myeloid leukemia cells expressing full-length PD-1H (C1498FF–PD-1H FL) and treated with anti–mPD-1H mAb (13F3) (B). Mice were assessed for in vivo leukemia proliferation using bioluminescence (C). A total of 200 μg of 13F3 or isotype control mAb was i.p. injected every 4 days from day 1 of transplantation of C1498FF–PD-1H cells (total 4 doses). Radiance indicates the mean value per group, and error bars represent SEM. P value determined by Student’s t test at each time point. n = 5. *P < 0.05. (D–F) In vivo growth of C1498FF–PD-1H s.c. tumor in B6 WT mice following anti–mPD-1H mAb treatment. A total of 200 μg of 13F3 or isotype control mAb was i.p. injected every 4 days from day 0 after s.c. injection of C1498FF–PD-1H cells (total 3 doses). (D) Tumor size was significantly smaller in the 13F3 treatment group compared with the isotype treatment group. Mean tumor volume ± SEM. Error bars represent SEM. n = 6 per group. *P < 0.05. (E and F) C1498FF–PD-1H s.c. tumor growth with 13F3 or isotype mAb treatment in B6 WT mice depleted of T cells or NK cells. n = 6. *P <0.05; ***P <0.01. P value determined by Student’s t test at each time point. (G) Immune cell subsets infiltrated in C1498FF–PD-1H tumors were assessed using mass cytometry. Left: percentages of granzyme B+ CD8+ T cells in total CD8+ T cells. Right: percentages of effector memory phenotype (CD44+CD62L–) CD8+ T cells in total CD8+ T cells. P value determined by Student’s t test. Error bars represent SEM. *P <0.05; **P <0.01.
Figure 5
Figure 5. Anti-human PD-1H mAb reverses immune evasion induced by human AML surface PD-1H.
(A) PD-1H suppressed T cell activation. Inhibition of polyclonal human T cells by human PD-1H on AML (HL-60). T cell proliferation was assessed by CFSE dilution. The diluted population was assessed by the percentage of total T cells. (B) The role of human AML PD-1H using a humanized mouse model. Human myeloid leukemia cells expressing PD-1H (HL-60–PD-1H) or not expressing PD-1H (HL-60-mock) were s.c. injected into NSG or NSG-S mice reconstituted with human peripheral blood mononuclear cells. Mice were sacrificed on day 14 and tumor tissues were removed to assess the size and to carry out IHC. (C and D) The volume of excised leukemia tumors (HL-60, left; MOLM14, middle; THP1, far right) expressing PD-1H or not expressing PD-1H or PD-1H–expressing leukemia tumors following anti-hPD-1H mAb treatment (n = 5 per group, *P < 0.05; **P < 0.01). P value determined by 1-way ANOVA (C) and Student’s t test (D). Mean tumor volume ± SEM. Error bars represent SEM. Photograph depicts HL-60 tumors removed from humanized NSG-S mice. (E) IHC of leukemia tumors expressing PD-1H following anti–hPD-1H mAb to assess CD4+ or CD8+ T cell infiltration (HL-60) and CD3 (MOLM14).
Figure 6
Figure 6. Mouse PD-1H blockade confers a synergistic antileukemic effect with mouse PD-1 blockade.
(A) Syngeneic mouse leukemia model using tail-vein injection with mouse myeloid leukemia cells expressing PD-1H (C1498FF–PD-1H) transplanted into B6 mice, which were then treated with anti–PD-1 and/or anti–PD-1H mAbs. Syngeneic mouse leukemia model using tail-vein injection with mouse myeloid leukemia cells not expressing PD-1H (C1498FF-mock) transplanted into WT B6 mice or PD-1H–KO mice, which were then assessed for in vivo antileukemia effect of genetic deletion of PD-1H in host mice with or without anti–PD-1 mAbs. (B) Synergistic antileukemia effect of anti–PD-1 mAb with anti–PD-1H mAb. In vivo proliferation was assessed by bioluminescence (left) and survival by a Kaplan-Meier plot (right). Radiance indicates the mean value per group, and error bars represent SEM. Data from 2 experiments were combined (n = 10). (C) Synergistic antileukemia effect of genetic deletion of PD-1H in host mice (PD-1H KO) with anti–PD-1 mAb. In vivo proliferation was assessed by bioluminescence (left) and survival by a Kaplan-Meier plot (right). Radiance indicates the mean value per group, and error bars represent SEM. Data from 2 experiments were combined (n = 10). (B and C) P value determined by simple linear regression method for statistical analysis of radiance and log-rank test for survival. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. These experiments were repeated 2 times. Repeated measures were determined by ANOVA with 2 factors (P > 0.05, no difference among experiments).
Figure 7
Figure 7. Human PD-1H blockade confers a synergistic antileukemic effect with human PD-1 blockade.
(A) A humanized AML mouse model to demonstrate a synergistic antileukemia effect of anti–hPD-1 with anti–hPD-1H mAbs. Human myeloid leukemia cells expressing PD-1H (THP1-WT) or not expressing PD-1H (THP1–PD-1H KO) were s.c. injected into NSG mice reconstituted with human peripheral blood mononuclear cells. (B) Tumor volume was assessed on days 2, 6, and 9. Anti–hPD-1 (100 μg) and/or anti–hPD-1H mAbs (100 μg) were injected on day 7. Day 9 tumor volume is represented. Mean tumor volume ± SEM. Error bars represent SEM. n = 5. *P < 0.05; ***P < 0.001; ****P < 0.001. P value determined by 1-way ANOVA.
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