Single-cell polyfunctional proteomics of CD4 cells from patients with AML predicts responses to anti–PD-1–based therapy

Acute myeloid leukemia (AML) remains a difficult disease to treat with a limited proportion of patients having long-term cures, despite several recently US Food and Drug Administration–approved therapies.¹  T cells can be used to eradicate leukemia as evident by grafted allogeneic T cells vs leukemia effected seen in postallogeneic stem cell transplantation.²  Harnessing the host T-cell activity against tumors via T-cell checkpoint inhibition has demonstrated significant success in many solid cancers and some lymphomas.³  Checkpoint blockade via PD-1, PD-L1, CTLA-4, and TIM-3 inhibition is being evaluated in several ongoing clinical trials in AML and myelodysplastic syndrome.⁴  In a phase 2 clinical trial in patients with relapsed/refractory (R/R) AML, combining the hypomethylating agent (HMA), azacitidine, with the PD-1 checkpoint inhibitor, nivolumab, demonstrated encouraging overall response rates of 33% (58% and 22% in HMA-naïve and HMA-pretreated patients), median event-free, and overall survival (OS), compared with a historical cohort of contemporary patients treated with azacitidine-based therapies, with an acceptable safety profile.⁵  Of note, patients who achieved complete response (CR)/incomplete blood count recovery (CRi)/partial response/hematologic improvement/stable disease had significantly improved OS compared with nonresponders (16.2 vs 4.1 months).⁵  However, unlike in solid tumors, long-term responses were rarely seen with AML. Patients with increased pretherapy bone marrow CD3⁺ or CD3⁺ CD8⁺ T cells were significantly more likely to achieve a response suggesting that biomarker-based strategies may further improve outcomes with checkpoint blockade in AML. However, bone marrow T-cell infiltration does not take into account the functional status of the T cells. In this study, we leveraged a multiplexed immune assay to assess the functional states of CD4⁺ and CD8⁺ cells at a single-cell level in pretherapy bone marrows in patients with R/R AML treated with azacitidine/nivolumab in the aforementioned clinical trial.

All patients were treated on protocol with azacitidine/nivolumab, as previously described (NCT02397720).⁵  This study was approved by MD Anderson institutional review board and conducted in accordance with the Declaration of Helsinki. Briefly, 11 nonresponders and 5 responders to treatment based on ELN2017 response criteria⁶  had available samples and were analyzed. Bone marrow samples were collected before and at end of second treatment cycle. Patient clinical and treatment characteristics are included in supplemental Table 1.

Detailed methods can be found in supplemental Material under “Single-cell 32-plex functional proteomic profiling.” Briefly, polyfunctional T cells co-secreting 2+ cytokines were assessed with the 32-plex proteomics (supplemental Table 2). The Polyfunctional Strength Index (PSI) of single T cells was computed as the percentage of polyfunctional cells, multiplied by the sum of the mean fluorescence intensity of the proteins secreted by those cells.^7-12 

A total of 16 patients with R/R AML (75% male), 81% with >1 salvage, median age 65 years (range, 47-90), who received a median of 3 (range, 2-17) cycles and a median of 6 (range, 3-33) doses of nivolumab had available samples for this analysis. Five of 16 (31.2%) patients achieved a CR/CRi on azacitidine/nivolumab, with a median time to CR/CRi of 4.07 (range, 0.9-12.6) months. The median OS of the 16 patients was 6 (range, 2.4-21.5) months, (responders vs nonresponders, P < .0001). The median duration of response was 5.2 (range, 0.5-14.7) months.

To identify predictors of response, we conducted single-cell polyfunctional assessment of CD4⁺ and CD8⁺ T cells in pretreatment bone marrows. We measured the degree of polyfunctionality and PSI, representing an aggregate of all single-cell multidimensional data into a single index, to determine any association with responses. The pretreatment bone marrow CD4⁺ but not CD8⁺ T cells had significantly higher frequency of polyfunctional cells (P = .04 and .15, respectively) and significantly higher PSI (P = .01 and .18, respectively) in patients who achieved CR/CRi compared with nonresponders (Figure 1A-B). Also, pretreatment bone marrow PSI of CD4⁺ (P < .001) but not CD8⁺ (P = .14) subsets demonstrated a significant positive association with OS across all patients, likely partly driven by response enrichment, suggesting that the degree of polyfunctional state of CD4⁺ cells may potentially also predict outcomes in patients with AML treated with azacitidine/nivolumab (Figure 1C). Further evaluation of the PSI composition across cell types revealed that interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) were the major drivers of enhanced PSI of pretherapy CD4⁺ subset, whereas Granzyme B, IFN-γ, MIP-1b, and TNF-α drove the nonsignificantly enhanced pretreatment PSI of CD8⁺ subset in the responders (Figure 1D). Although we do not have further subtyping of CD4⁺ cells, the increased IFN-γ and TNF-α pattern is generally distinctive for the CD4⁺ T_H1 functional state.¹³ 

Although checkpoint blockade-based responses in solid tumors are generally thought to be driven by CD8⁺ cells, recent studies suggest that CD4⁺ cells are critical in initiating and sustaining an immunotherapy response.^14,15  Further, CD4⁺ cells can independently recognize mutant neoepitopes,¹⁶  tumor infiltrating T_H1-like CD4⁺ T cells can acquire antitumor cytotoxic activity,¹⁷  and CD4⁺ rather than CD8⁺ T cells are critical for inducing responses to neoantigen vaccination in melanoma.¹⁸  Collectively, these studies support a significant role for CD4⁺ in induction of antitumor immunotherapy-based responses, which is consistent with our findings in AML.

We next applied 3-dimensional t-distributed stochastic neighbor embedding^19-21  to visualize high-dimensional data in low-dimensional space to stratify functional cell clustering with distinct cytokine secretion patterns between patient groups (Figure 2A). Our findings revealed that CD4⁺ and CD8⁺ cells from responders and nonresponders had distinct clustering patterns (Figure 2A, left). However, responders demonstrated a more prominent increase in polyfunctional cell subsets with antitumor-associated proteins in CD4⁺ and CD8⁺ subsets (purple circles), compared with nonresponders (Figure 2A, middle and left). When clustered by degree of polyfunctionality, highly polyfunctional groups clustered together in both CD4⁺ and CD8⁺ T cells (Figure 2A, middle). Of note, the most dominant functional group across CD4⁺ and CD8⁺ cells was the effector protein pattern, which was more notable in the pretreatment bone marrows of responders compared with nonresponders (Figure 2A, left and right) and also corresponded to the highly polyfunctional group. These findings suggest that an effector functional phenotype is the predominant CD4⁺ and CD8⁺ T-cell phenotype in pretreatment bone marrows of patients with AML who responded to azacitidine/nivolumab. In addition, we used a functional heatmap to compare the frequency of various expressed monofunctional and polyfunctional groups.^7,19,21,22  Compared with nonresponders, the pretherapy CD4⁺ T cells of responders had more distinct and unique polyfunctional groups (green rectangles) such as coexpression of TNF-α/IFN-γ/interleukin-8, TNF-α/IFN-γ/Granzyme B or TNF-α/IFN-γ/Perforin/interleukin-8 proteins (Figure 2B). However, CD8⁺ T cells did not show such distinct patterns between responders and nonresponders. These observations are consistent with the pretherapy PSI patterns noted in CD4⁺ T cells but not CD8⁺ T cells of responders, further highlighting the association of bone marrow polyfunctional CD4⁺ subset with response to azacitidine/nivolumab therapy.

The single-cell multiplexed functional proteomics precision profiling demonstrated that the pretreatment PSI of CD4⁺ cells was significantly associated with response and OS in relapsed patients treated with azacitidine/nivolumab and should be evaluated prospectively to ascertain whether this could be an effective biomarker to select AML patients for PD-1–based therapies. Pretreatment PSI should be evaluated in association with treatment outcomes with CTLA4, PD-L1, TIM3, and other T-cell based strategies in clinical trials in AML and may have a potential role as a biomarker in the wider sphere of immunotherapy in AML. Such analyses are warranted and encouraged.

This work was supported in part by the MD Anderson Cancer Centre Support Grant CA016672, the MD Anderson Cancer Center Leukemia SPORE CA100632, the Charif Souki Cancer Research Fund, the Dick Clark Immunotherapy Fund, and generous philanthropic contributions to the MD Anderson Moon Shots Program.

Contribution: H.A.A., S.M., M.C., J.Z., M. Andreeff, M.K., and N.D. designed the study, analyzed the data, and wrote the paper; H.A.A., J.M., G.C.I., M. Alfayez, S.M., M.C., and J.Z. collected and analyzed the data; G.C.I. and M. Andreeff performed the molecular and cytogenetic analysis; N.D., M. Andreeff, E.J., S.M.K., M.K., and G.G.-M. enrolled patients; and all authors contributed to data collection, reviewed and approved the manuscript, and shared final responsibility for the decision to submit.

Conflict-of-interest disclosure: M.K. reports grants and other from AbbVie, grants and other from Genentech, grants and other from F. Hoffman La-Roche, grants and other from Stemline Therapeutics, other from Amgen, grants and other from Forty-Seven, other from Kisoji, grants from Eli Lilly, grants from Cellectis, grants from Calithera, grants from Ablynx, grants from Agios, grants from Ascentage, grants from Astra Zeneca, other from Reata Pharmaceutical, grants from Rafael Pharmaceutical, grants from Sanofi, outside the submitted work, and a patent US 7 795 305 B2 CDDO-compounds and combination therapies with royalties paid to Reata Pharm., a patent Combination Therapy with a mutant IDH1 inhibitor and a BCL-2 licensed to Eli Lilly, and a patent 62/993 166 combination of a MCL-1 inhibitor and midostaurin, uses and pharmaceutical compositions thereof pending to Novartis. E.J. reports research grants and consultancy from Abbvie, Adaptive Biotechnology, Amgen, BMS, Pfizer, and Takeda. N.D. reports research funding from Daiichi Sankyo, Bristol-Myers Squibb, Pfizer, Karyopharm, Sevier, Genentech, Astellas, Abbvie, Genentech, Novimmune, Amgen, Trovagene, Gilead, FATE Therapeutics, Trillium, Hanmi, Newave, Glycomimetics, and ImmunoGen; he has served in a consulting or advisory role for Daiichi Sankyo, Bristol-Myers Squibb, Pfizer, Novartis, Celgene, AbbVie, Genentech, Servier, Trillium, Syndax, Trovagene, Astellas, Gilead, STAR Therapeutics, KITE, and Agios. M. Andreeff has received grant or research support from Daiichi-Sankyo, Breast Cancer Research Foundation, ONO Pharmaceuticals, Karyopharm, Amgen, AstraZeneca, and Oxford Biomedica UK; paid consultancy from Daiichi-Sankyo, Aptose, Senti-Bio, Glycomimetics, Syndax, Medicxi; membership on advisory committees or review panels, board membership in Cancer UK, Leukemia & Lymphoma Society, German Research Council, NCI-RDCRN (Rare Disease Clinical Network), CLL Foundation, Novartis; and ownership interest (eg, stocks, stock options) in Reata, Aptose, Eutropics, SentiBio, Chimerix, and Oncolyze. S.M., M.C., and J.Z. are employed by and have equity ownership in IsoPlexis.

Correspondence: Naval Daver, Department of Leukemia, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 0428, Houston, TX 77030; e-mail: ndaver@mdanderson.org.