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Dual CD4-based CAR T cells with distinct costimulatory domains mitigate HIV pathogenesis in vivo

Nature Medicine volume 26, pages 1776–1787 (2020)Cite this article

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Abstract

An effective strategy to cure HIV will likely require a potent and sustained antiviral T cell response. Here we explored the utility of chimeric antigen receptor (CAR) T cells, expressing the CD4 ectodomain to confer specificity for the HIV envelope, to mitigate HIV-induced pathogenesis in bone marrow, liver, thymus (BLT) humanized mice. CAR T cells expressing the 4-1BB/CD3-ζ endodomain were insufficient to prevent viral rebound and CD4+ T cell loss after the discontinuation of antiretroviral therapy. Through iterative improvements to the CAR T cell product, we developed Dual-CAR T cells that simultaneously expressed both 4-1BB/CD3-ζ and CD28/CD3-ζ endodomains. Dual-CAR T cells exhibited expansion kinetics that exceeded 4-1BB-, CD28- and third-generation costimulated CAR T cells, elicited effector functions equivalent to CD28-costimulated CAR T cells and prevented HIV-induced CD4+ T cell loss despite persistent viremia. Moreover, when Dual-CAR T cells were protected from HIV infection through expression of the C34-CXCR4 fusion inhibitor, these cells significantly reduced acute-phase viremia, as well as accelerated HIV suppression in the presence of antiretroviral therapy and reduced tissue viral burden. Collectively, these studies demonstrate the enhanced therapeutic potency of a novel Dual-CAR T cell product with the potential to effectively treat HIV infection.

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Fig. 1: BLT mouse-derived HIV-specific CAR T cells are functionally indistinguishable from human-derived CAR T cells in vitro.
Fig. 2: CAR T cells expressing the 4-1BB costimulatory domain exhibit a proliferative advantage and induce B cell aplasia in vivo.
Fig. 3: HIV-specific CAR.BBζ T cells display features of T cell exhaustion after failing to control viral rebound.
Fig. 4: Dual-CAR TCP mitigates CD4+ T cell loss and exhibits superior proliferative capacity.
Fig. 5: HIV-resistant Dual-CAR T cells mediate superior virus-specific immune responses.
Fig. 6: Mitigating CAR T cell infection improves control over HIV replication.

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Data availability

All of the construct sequences described in this article are published (see ref. 26). All materials described in this manuscript are available via a material transfer agreement with the University of Pennsylvania or the Ragon Institute. All data are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

We thank C. Ellebrecht and A. Payne (University of Pennsylvania) for the HIVYU2 Envelope-transduced K562 cell line. We thank W. Garcia-Beltran for artistic contributions. This study was supported by NIH grants P01HL129903 (T.M.A.), U19AI117950 (J.L.R.) and UM1AI126620 (J.L.R.) funded by NIAID, NIDA, NIMH and NINDS; T32 grant AI007632 (C.R.M.); and an NIAID F32 grant AI136750 (D.T.C.). We also acknowledge support from the Ragon Institute of MGH, MIT and Harvard and the MGH Scholars program (T.M.A.). We thank the Human Immune System Mouse Core at the Ragon Institute of MGH, MIT and Harvard and the Stem Cell and Xenograft Core at the University of Pennsylvania for the generation of BLT humanized mice, the Penn Center for AIDS Research (P30-AI045008) for purified human T cells, and the Allen and Riley laboratories for helpful comments.

Author information

Author notes

  1. These authors contributed equally: Colby R. Maldini, Daniel T. Claiborne.

  2. These authors jointly supervised this work: James L. Riley, Todd M. Allen.

Authors and Affiliations

  1. Department of Microbiology, Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Colby R. Maldini & James L. Riley

  2. Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA

    Daniel T. Claiborne, Ken Okawa, Tao Chen, Karen A. Power, Radiana T. Trifonova, Katharine Krupp, Meredith Phelps, Vladimir D. Vrbanac, Serah Tanno, Timothy Bateson, Christian L. Boutwell & Todd M. Allen

  3. Department of Pathology & Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Derrick L. Dopkin & Xiaochuan Shan

  4. Human Immune System Mouse Program, Massachusetts General Hospital, Boston, MA, USA

    Vladimir D. Vrbanac & Serah Tanno

  5. Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    George J. Leslie & James A. Hoxie

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Contributions

C.R.M., D.T.C., K.O., C.L.B., J.L.R. and T.M.A. conceived and designed the project and contributed to the interpretation of data. C.R.M., D.T.C., K.O., T.C., D.L.D., X.S., K.A.P., R.T.T., K.K., M.P., V.D.V., S.T., T.B., G.J.L. and J.A.H. contributed to the acquisition and analysis of data. C.R.M., D.T.C., C.L.B., J.L.R. and T.M.A. drafted the manuscript.

Corresponding authors

Correspondence to James L. Riley or Todd M. Allen.

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Competing interests

C.R.M. and J.L.R. have filed an institution-owned patent (20180265565: Method of Redirecting T Cells to Treat HIV Infection) describing the construction of these HIV-specific CARs specific to Figs. 1 and 2. This patent application has been published but has not yet been granted. C.R.M. and J.L.R. have also filed an institution-owned patent (Dual CAR Expressing T Cells Individually Linked to CD28 and 4-1BB) specific to Figs. 4–6. G.J.L., J.A.H. and J.L.R. have also filed an institution-owned patent (Non-Signaling HIV Fusion Inhibitors And Methods Of Use Thereof) specific to Figs. 5 and 6. J.L.R. cofounded a company called Tmunity Therapeutics that has the rights to license the technology described in this paper. J.L.R. holds an equity interest in Tmunity. C.R.M. and J.L.R. declare no other competing financial interests. No other authors declare any competing financial interests. No authors declare any nonfinancial interests.

Additional information

Peer review information Alison Farrell was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 CD28 costimulation enhances the ex vivo effector function of CAR T cells.

HIV-uninfected mice were infused with an equal mixture of CD4-based CAR T cells expressing either CD3-ζ, 4-1BB/CD3-ζ and CD28/CD3-ζ costimulatory domains linked to unique fluorescent proteins to facilitate identification in vivo as described in Fig. 2 legend. Cumulative data indicating the frequency of TNF+, IL-2+ and MIP-1β+ CAR.BBζ and CAR.28ζ T cells within the same mice after ex vivo stimulation with K.Env (+) or K.WT (-) cells. Data represents the aggregate of cytokine producing cells from liver and terminal blood (n = 8). CAR.ζ T cells were too infrequent for analysis. Data shows box and whisker plots where the middle line indicates median, bounds of the box show 25th to 75th percentiles, and bars extend to min and max values. Symbols represent biologically independent animals. Significance was calculated using two-sided Wilcoxon matched-pairs signed rank test.

Source data

Extended Data Fig. 2 CAR.BBζ T cells accumulate multiple inhibitory receptors as disease progresses.

a, Frequency of CD4+ and (b) CD8+ CAR.BBζ T cells (G1; n = 6) and CAR.BBΔζ T cells (G2; n = 6) co-expressing TIGIT and PD-1 after infusion. Shaded box indicates the window of ART. Symbols and error bars indicate mean ± SEM. c, Frequency of CD4+ and (d) CD8+ CAR.BBζ T cells (G1) and CAR.BBΔζ T cells (G2) co-expressing TIGIT, PD-1 and 2B4 in tissues 12 weeks post-infusion. e, Cumulative data indicating the frequency of 2B4+, PD-1+ and TIGIT+ CD4+ CAR.BBζ T cells (G1) compared to CAR- CD4+ T cells (G1) within the spleens of the same mice, and (f) CD8+ CAR.BBζ T cells (G1) compared to CAR- CD8+ T cells (G1) within the spleens of the same mice. c–f, Bars indicate mean, error bars show ± SEM and symbols represent individual mice. Significance was calculated using two-sided Wilcoxon rank-sum test. Sample sizes in these studies represent biologically independent animals.

Source data

Extended Data Fig. 3 EomeshiT-betdim CAR.BBζ T cells accumulate from acute to chronic phases of infection.

BLT mice were infected with HIVJRCSF and infused 48 h later with either 2×107 CAR.BBζ T cells (n = 5) or inactive control CAR.BBΔζ T cells (n = 3). a, FACS plots show the change in Eomes and T-bet expression within the different CAR T cell types over time. b, Summary data indicating the longitudinal frequency of EomeshiT-betdim CD8+ (left panel) and CD4+ (right panel) CAR T cells (left y-axis), and mean log plasma HIV RNA (copies mL−1) (right y-axis). Thin dotted line denotes limit of viral load quantification. Symbols and error bars indicate mean ± SEM. c, Spearman correlation analysis of frequency of EomeshiT-betdim CD8+ CAR.BBζ T cells compared with viral burden measured as the frequency of HIVGAG+ CD8- T cells in various tissues 10 weeks post-infection. Sample sizes in these studies indicate biologically independent animals.

Source data

Extended Data Fig. 4 Dual-CAR T cell product transiently delays CD4+ T cell loss despite persistent HIVJRCSF infection.

BLT mice received Dual-CAR T cell product (TCP) (n = 6) 48 h after HIVJRCSF challenge, while control mice were untreated (Untx) (n = 5). a, Concentration of peripheral total memory CD4+ T cells (CAR−). b, Concentration of peripheral central memory (CD45RA−CD27+CCR7+; left panel), transitional memory (CD45RA-CD27+CCR7−; middle panel), and effector memory (CD45RA-CD27-CCR7-; right panel) CD4+ T cells (CAR-). c, Frequency of memory CD4+ T cell (CAR-) subsets in tissues 8 weeks post-infection. a, b, Significance was calculated using a two-sided Wilcoxon rank-sum test. Symbols and bars indicate mean, and error bars show ± SEM. Sample sizes indicate biologically independent animals.

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Extended Data Fig. 5 Dual-CAR T cell product prevents CD4+ T cell loss despite persistent HIVMJ4 infection.

BLT mice were infused with Dual-CAR T cell product (TCP) (n = 6) 48 h post-HIVMJ4 challenge, while control mice were untreated (Untx) (n = 6). a, Concentration of peripheral total memory CD4+ T cells (CAR−). b, Concentration of peripheral central memory (CD45RA-CD27+CCR7+; right panel), transitional memory (CD45RA−CD27+CCR7-; middle panel), and effector memory (CD45RA-CD27-CCR7-; left panel) CD4+ T cells (CAR-). c, Frequency of memory CD4+ T cell (CAR-) subsets in tissues 8 weeks post-infection. a, b, Significance was calculated using a two-sided Wilcoxon rank-sum test. Symbols and bars indicate mean, while error bars show ± SEM. Sample sizes indicate biologically independent animals.

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Extended Data Fig. 6 Dual-CAR T cells exhibit superior in vivo expansion compared to 4-1BB-costimulated, CD28-costimulated, and 3rd-generation CAR T cells.

a, BLT mice were challenged with either HIVJRCSF (n = 6) or HIVMJ4 (n = 6) and infused with 2×107 Dual-CAR T cell product (TCP). Fold-change in CAR T cell concentration from baseline to peak levels in peripheral blood. Data is the aggregate of both infection cohorts. b, Schematic shows the structural components of the 3rd-generation (3 G) CD4-based CAR construct. c–e, Dual-CAR T cell product and 3G-CAR T cells were combined at an equal frequency prior to infusion into uninfected mice (n = 9). c, FACS plots indicate the frequency of Dual-CAR and 3G-CAR T cells present within the pre-infusion T cell product. d, Longitudinal concentration of peripheral CAR T cells following adoptive transfer into HIV-negative mice. Symbols and error bars indicate mean ± SEM. e, At 2 weeks post-infusion, mice received either 107 irradiated K.Env cells (n = 6) or 107 irradiated K.WT cells (n = 3). Fold change in the concentration of peripheral CAR T cells 1-week post-K562 infusion from baseline concentration prior to K562 infusion. a, e, Bar and error bars indicate mean ± SEM, and symbols represent individual mice. a, d, e, Two-sided Wilcoxon rank-sum test was used to calculate significance. Sample sizes in these studies indicate biologically independent animals.

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Extended Data Fig. 7 C34-CXCR4+ CAR T cells are selected for during chronic infection and exhibit superior ex vivo effector functions.

a, BLT mice were infected with HIVJRCSF and 48 h later infused with 107 C34-CXCR4+ Dual-CAR T cell product (TCP). FACS plots indicate the frequency of C34-CXCR4+ throughout infection. b, Mice were infected with HIVMJ4 and 48 h later were infused with 106 C34-CXCR4+ CAR.BBζ (n = 5), CAR.28ζ (n = 5), or purified Dual-CAR (n = 4) T cells. Frequency of C34-CXCR4+ CAR T cells in tissue 8 weeks post-infection. Thin dotted line indicates the frequency of C34-CXCR4+ CAR T cells in the pre-infusion TCP for the indicated CAR T cell type. Line and error bars indicate mean ± SEM. c, d, Mice were infected with HIVMJ4 and 48 h later received 106 C34-CXCR4+, purified CAR.BBζ.BBζ (n = 3), CAR.28ζ.28ζ (n = 4), or Dual-CAR (n = 3) T cells. c, FACS plots and (d) cumulative data show the frequency of each CD8+ CAR T cell population expressing MIP-1β and CD107a, and the frequency of CAR T cells with cytotoxic potential (granzyme B+ perforin+ CD107a+). CAR T cells were isolated from the spleen and bone marrow of mice 8 weeks post-infection and ex vivo stimulated. Significance was calculated using two-sided Wilcoxon matched-pairs signed rank test. For all data, symbols represent individual mice. Sample sizes in these studies indicate biologically independent animals.

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Extended Data Fig. 8 CAR T cells from HIV-infected mice exhibit ex vivo cytotoxic function.

HIVJRCSF-infected mice (n = 3) treated with the Dual-CAR TCP were euthanized and the bone marrow cells were ex vivo stimulated with K.Env or K.WT cells for 24 h at the indicated E:T ratios. a, Representative FACS plots and (b) cumulative data shows the induction of active caspase-3 within the different target cell populations. Symbols and error bars indicate mean ± SEM. Sample size indicates biologically independent animals.

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Extended Data Fig. 9 Dual-CAR and CAR.28ζ T cells exhibit similar ex vivo functional profiles.

BLT mice were challenged with HIVJRCSF (n = 5) and infused with 2×107 Dual-CAR T cell product (TCP) 48-hours post infection. a, Frequency of CD8+ and (b) CD4+ CAR T cell populations from tissue at necropsy (8-weeks post-infection) within the same mice expressing CD107a, MIP-1β, IL-2 and TNF after ex vivo stimulation. Bars and error bars indicate mean ± SEM, and symbols represent individual mice. Significance was calculated using two-sided Wilcoxon rank-sum test. c, Principle Components Analysis (PCA) of IL-2, TNF, MIP-1β, and CD107a expression in ex vivo stimulated CD8+ and CD4+ CAR T cells from PBMCs of HIVJRCSF-infected mice (n = 5). Sample sizes in these studies represent biologically independent animals.

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Extended Data Fig. 10 HIV-resistant Dual-CAR TCP reduces virus replication in vivo.

a, Frequency of HIVGAG+ CD8- T cells (CAR-) within the bone marrow and spleen of HIVJRCSF-infected mice (n = 6) and (b) HIVMJ4-infected mice (n = 6) that were treated 48 h post-challenge with the Dual-CAR T cell product (TCP) or were untreated (Untx). c, Mean log plasma HIVMJ4 RNA (copies mL−1) after ART discontinuation of mice infused at ART initiation with 107 protected >98% C34-CXCR4+ (n = 5) or partially-protected <20% C34-CXCR4+ (n = 7) Dual-CAR TCP, or were untreated (n = 9). d, e, HIVBAL-infected mice were ART-treated and simultaneously infused with 107 HIV-resistant Dual-CAR TCP (n = 6) or were untreated (n = 6). d, Mean log plasma HIV RNA (copies mL−1). Shaded box indicates ART and arrow indicates CAR TCP infusion. e, Percent log reduction in plasma HIV RNA from pre-ART (week 3) and 0.5 and 1 week post-ART. For all data, bars and error bars indicate mean ± SEM, and symbols represent individual mice. Significance was calculated for (a–d) by two-sided Wilcoxon rank-sum test and (e) two-sided Kolmogorov-Smirnov test. Sample sizes in these studies indicate biologically independent animals.

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Maldini, C.R., Claiborne, D.T., Okawa, K. et al. Dual CD4-based CAR T cells with distinct costimulatory domains mitigate HIV pathogenesis in vivo. Nat Med 26, 1776–1787 (2020). https://doi.org/10.1038/s41591-020-1039-5

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