Comments for:
Abstract
TGF-β plays a critical role in maintaining immune cells in a resting state by inhibiting cell activation and proliferation. Resting HIV-1 target cells represent the main cellular reservoir after long-term antiretroviral therapy (ART). We hypothesized that releasing cells from TGF-β–driven signaling would promote latency reversal. To test our hypothesis, we compared HIV-1 latency models with and without TGF-β and a TGF-β type 1 receptor inhibitor, galunisertib. We tested the effect of galunisertib in SIV-infected, ART-treated macaques by monitoring SIV-env expression via PET/CT using the 64Cu-DOTA-F(ab′)2 p7D3 probe, along with plasma and tissue viral loads (VLs). Exogenous TGF-β reduced HIV-1 reactivation in U1 and ACH-2 models. Galunisertib increased HIV-1 latency reversal ex vivo and in PBMCs from HIV-1–infected, ART-treated, aviremic donors. In vivo, oral galunisertib promoted increased total standardized uptake values in PET/CT images in gut and lymph nodes of 5 out of 7 aviremic, long-term ART-treated, SIV-infected macaques. This increase correlated with an increase in SIV RNA in the gut. Two of the 7 animals also exhibited increases in plasma VLs. Higher anti-SIV T cell responses and antibody titers were detected after galunisertib treatment. In summary, our data suggest that blocking TGF-β signaling simultaneously increases retroviral reactivation events and enhances anti-SIV immune responses.
Authors
Sadia Samer, Yanique Thomas, Mariluz Araínga, Crystal Carter, Lisa M. Shirreff, Muhammad S. Arif, Juan M. Avita, Ines Frank, Michael D. McRaven, Christopher T. Thuruthiyil, Veli B. Heybeli, Meegan R. Anderson, Benjamin Owen, Arsen Gaisin, Deepanwita Bose, Lacy M. Simons, Judd F. Hultquist, James Arthos, Claudia Cicala, Irini Sereti, Philip J. Santangelo, Ramon Lorenzo-Redondo, Thomas J. Hope, Francois J. Villinger, Elena Martinelli
Comment on Samer et al.
Submitter: Michele Di Mascio | mdimascio@niaid.nih.gov
Authors: Sharat Srinivasula, Jorge A. Carrasquillo
National Institute of Allergy and Infectious Diseases
Published June 28, 2023
Dear Editor,
We read with enthusiasm the paper by Samer et al., however, we are concerned about the interpretations of some of the PET images, with notable implications that may invalidate some of the conclusions of the study.
Figure 3H shows that the highest increases in lymph node (LN) uptake are in monkeys A14X060, A14X064, and A14X013. In these animals, PET images show clear evidence of significant extravasation of the injected 89Zr-DFO-antibody into the subcutaneous tissues ipsilateral to the high LN uptake. A fourth animal (A14X027) has a slight increase in LN uptake with smaller extravasation. It is well known that when radiolabeled antibodies are intentionally or unintentionally (infiltration) administered by subcutaneous or intradermal route, they find their way into the lymphatics and then non-specifically concentrate in regional draining nodes (1), as it appears to be the case for the animals listed above since the radiotracer uptake in LNs was much higher on the side of infiltration than the contralateral side or than in distant nodes. For this reason, the SUV analysis should not have included those LNs in Figure 3H. Excluding those uptakes, we believe the MIP movies in the supplemental materials clearly highlight the lack of increase in uptake in nodal regions following galunisertib administration and point to important sensitivity limitations of the in-vivo imaging system presented in the study, while simultaneously questioning the feasibility of non-invasively imaging low levels of viral replication using the 7D3 radiolabeled mAb, as previously reported by the same imaging-team co-authors in two previous publications (2, 3) and presented in this research article as a validated methodology.
Consistent with the above concern, we believe that it is unlikely that an LRA administration (galunisertib) can induce such dramatic heterogeneity in gp120 expression among different clusters of LNs or in different segments of the bowel with discordant changes. It is also surprising that evidence of specific binding would be visualized in clusters of LNs and not the spleen, given that these two compartments show similar levels of cell-associated RNA viral load (4, 5) or gp120 (6), and that the LNs but not the spleen suffer from partial volume effect.
Additionally, given that the increases in SIV-RNA levels in the gut and LNs were similar (from Figure 4A), if our interpretation of the mechanism of enhanced LNs radiotracer uptake (based on the published images) is correct, then the increase in bowel uptake likely reflects a well-known phenomenon of non-specific intraluminal uptake of the radiolabeled antibody, hence necessitating an ex-vivo probe-binding capacity validation (e.g. in primary cells or autoradiography tissue section analysis). We suspect that in contrast to other studies that attempted to image lentiviral replication with radiolabeled anti-envelope antibodies, this study did not exclude in the analysis areas that are consistent with the intraluminal antibody excretion in bowel segments (7).
Michele Di Mascio
AIDS Imaging Research Section, Division of Clinical Research, NIAID, NIH, Bethesda, MD, 20892, USA
Sharat Srinivasula
AIDS Imaging Research Section, Clinical Monitoring Research Program Directorate, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA
Jorge A. Carrasquillo
Molecular Imaging and Therapy Service, Radiology Department, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.Molecular Imaging Branch, Center for Cancer Research, NCI, NIH, Bethesda, MD, 20892, USA
References:
1. Keenan AM, Weinstein JN, Carrasquillo JA, Bunn PA, Jr., Reynolds JC, Foon KA, et al. Immunolymphoscintigraphy and the dose dependence of 111In-labeled T101 monoclonal antibody in patients with cutaneous T-cell lymphoma. Cancer Res. 1987;47(22):6093-9.
2. Santangelo PJ, Rogers KA, Zurla C, Blanchard EL, Gumber S, Strait K, et al. Whole-body immunoPET reveals active SIV dynamics in viremic and antiretroviral therapy-treated macaques. Nature methods. 2015;12(5):427-32.
3. Santangelo PJ, Cicala C, Byrareddy SN, Ortiz KT, Little D, Lindsay KE, et al. Early treatment of SIV+ macaques with an alpha(4)beta(7) mAb alters virus distribution and preserves CD4(+) T cells in later stages of infection. Mucosal Immunol. 2018;11(3):932-46.
4. Horiike M, Iwami S, Kodama M, Sato A, Watanabe Y, Yasui M, et al. Lymph nodes harbor viral reservoirs that cause rebound of plasma viremia in SIV-infected macaques upon cessation of combined antiretroviral therapy. Virology. 2012;423(2):107-18.
5. Pahar B, Kuebler D, Rasmussen T, Wang X, Srivastav SK, Das A, et al. Quantification of Viral RNA and DNA Positive Cells in Tissues From Simian Immunodeficiency Virus/Simian Human Immunodeficiency Virus Infected Controller and Progressor Rhesus Macaques. Front Microbiol. 2019;10:2933.
6. Santosuosso M, Righi E, Lindstrom V, Leblanc PR, and Poznansky MC. HIV-1 envelope protein gp120 is present at high concentrations in secondary lymphoid organs of individuals with chronic HIV-1 infection. J Infect Dis. 2009;200(7):1050-3.
7. Beckford-Vera DR, Flavell RR, Seo Y, Martinez-Ortiz E, Aslam M, Thanh C, et al. First-in-human immunoPET imaging of HIV-1 infection using (89)Zr-labeled VRC01 broadly neutralizing antibody. Nat Commun. 2022;13(1):1219.
Reply to Di Mascio et al.
Submitter: Elena Martinelli | elena.martinelli@northwestern.edu
Authors: Elena Martinelli, and Francois Villinger
Published June 28, 2023
We thank Di Mascio and colleagues, for their interest in our study. We should first point out that we used we used 64Cu-DOTA-F(ab')2, rather than a 89Zr-DFO-antibody. Second, the probe was administered IV via a catheter to ensure that there would be no or minimal spillover in tissues adjoining the injection site. We acknowledge that the subcutaneous signals in two animals (A14X060 and A14X064) could explain, in part, the signal in the ipsilateral nodes. However, in these 2 animals, there was a large increase in plasma VL following Galunisertib, which more than proves viral reactivation. A14X013 had no tissue signal at week 2 and A14X027 had no increase in signal in nodes. Third, any interpretation of signals in nodes is irrelevant to the signals we observed in gut tissue. The correlation between vRNA and PET in the gut tissues in Fig 4B, and the correlation between PET signals and vRNA in the gut resections in Fig S9 proves that the PET signal in the gut represents sites of viral reactivation. Additional microscopy images of vRNA by RNAscope at the site of PET signal following the post-Gal gut resection were presented at the workshop on HIV persistence during therapy in Miami in 2023. These data add to previously published data1,2 as well as data presented by Dr. Hope’s group in other monkeys where the PET signal was used to perform PET-guided necropsies to document site of viral replication by immunofluorescence and electron microscopy particularly in the gut. Finally, while not conducted in this study, combining fluorescent and 64Cu-labeled probes administered in vivo has shown that binding of our 7D3-64Cu-DOTA-F(ab')2 probe is specific in vivo (in preparation).
While we appreciate the various hypotheses advanced here, our approach is to report what we see from the animal experiment. SIV infection is not uniform across the lymphatic system and in tissues. As reported in our 2016 Nature article1, the gut can present >4 logs of vRNA level variations depending on which segment is evaluated. Hence, HIV replication and especially stochastic reactivation events are highly compartmentalized in tissues. This explains well the “dramatic heterogeneity in gp120 expression among different clusters of LNs or in different segments of bowel” noted by the authors of the comment. Of note, the in vivo SIV latency reversal following TGFb blockade, including the increase in PET-signal, was recapitulated in follow-up studies (HIV persistence workshop Miami 2022 and manuscript in preparation).
Elena Martinelli
Cell and Developmental Biology, Northwestern University, 60611, Chicago, IL, USA
Francois Villinger
New Iberia Research Center, University of Louisiana at Lafayette, 70562, New Iberia, LA, USA
References
1. Santangelo PJ, Rogers KA, Zurla C, et al. Whole-body immunoPET reveals active SIV dynamics in viremic and antiretroviral therapy-treated macaques. Nat Methods 2015;12(5):427-32, doi:10.1038/nmeth.3320
2. Santangelo PJ, Cicala C, Byrareddy SN, et al. Early treatment of SIV+ macaques with an alpha4beta7 mAb alters virus distribution and preserves CD4(+) T cells in later stages of infection. Mucosal immunology 2017, doi:10.1038/mi.2017.112
