Immune Checkpoint Molecules and Glucose Metabolism in HIV-Induced T Cell Exhaustion

Round 1

Reviewer 1 Report

I feel this is a nice review regarding immune checkpoint molecules and glucose metabolism in HIV-infected T cell and its exhaustion. Two figures are fine and the text is written well.  In addition, the possibilities of therapy on the view point of this review are also described at the end. Thus, this manuscript possesses no problems.

Author Response

We thank the reviewer for your time and effort that you have dedicated to providing valuable feedback on our manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

This is an interesting review, regarding immune checkpoint expression and glucose metabolism in HIV infection. Its main limitation is that the authors did not dig in depth in most of the sections of the review, that should be expanded.

Revisions:

·        Most of the manuscript is cantered in T cell metabolism in HIV, I would change “Immune checkpoint molecules and glucose metabolism” for “T cell metabolism” in the title.

·        The abstract states that the review highlights the interplay between immune checkpoint signalling pathway and glucose metabolism, however, only ICIs expression are considered in the review. This should be rewritten to “immune checkpoint molecules”, or otherwise the different immune checkpoint signalling pathways should be explained in the manuscript.

·        TIM3 and GITR among other immune checkpoint molecules should also be discussed.

·        For some ICIs, only a sentence of their role on HIV is stated. The ICIs expression and role on HIV should be further discussed for all ICIs (section 2 should be expanded).

·        Very little information is presented on the 3.1, 3.2 and 3.3 sections regarding its role in HIV. All the sections can be expanded and oriented to HIV. This could serve as an introduction part of the review.

·        Sections 4.2, 4.2 and 4.4 could be further developed with more bibliographical data.

·        Section 5 should be presented as a discussion. Also, preclinical or clinical data of ICIs inhibition in the context of HIV should be discussed.

Author Response

We gratefully thank the reviewer for providing valuable and insightful comments to improve our manuscript. We have carefully addressed all the comments raised.

1. We have revised the title from "Immune Checkpoint Molecules and Glucose Metabolism in HIV-induced T Cell Exhaustion", to " Immune Checkpoint Molecules and T Cell Metabolism in HIV Infection".

2. Line 26-29, Abstract: We have replaced the “immune checkpoint signaling pathways” with “immune checkpoint molecules”, as suggested.

3. Line 179-202; 2.3. TIM-3 : We have added the information on TIM3 to the manuscript, we do not include GITR due to limited data available. Added text are as follow:

"TIM-3 is initially identified as a Th1-specific transmembrane protein that characterizes the differentiated Th1 CD4⁺ T cell [49]. TIM-3 regulates T cell proliferation, production of pro-inflammatory cytokines IFN-g and peripheral tolerance [50], which are driven by the binding of galectin-9, a ligand of Tim-3 [51]. The TIM-3/galectin-9 pathway triggers inhibitory signaling, reduces IFN-g producing Th1 cells and induces cell death in activated Th1 cells [51].

During HIV infection, viral proteins such as Nef and Vpu exert opposing effects on TIM-3 expression level in infected CD4⁺ T cells. As shown by a recent in vitro study, HIV-1 Nef protein mediates the upregulation of TIM-3 through its dileucine motif and contradictorily, activates the infected cells through TCR signaling [52]. Conversely, Vpu protein downregulates TIM-3 surface expression on infected CD4⁺ T cells via its transmembrane domain and alters its subcellular localization [53]. These effects might because of the early expression of Nef to favor viral replication, while Vpu is expressed late to facilitate viral release.

In progressive HIV infection, high expression of TIM-3 on HIV-1-specific CD8⁺ T cell is correlated with the frequency of dysfunctional T cell population [15]. Regulatory T cell utilizes the TIM-3/galectin-9 pathway to suppress proliferation of HIV-specific CD8⁺ T cell; the protective allele HLA-B*27- and HLA-B*5-restricted CD8⁺ T cells present low level of TIM-3 upregulation can evade the regulatory T cell-mediated suppression [54]. This explains why HIV elite controllers possessing these HLA alleles are able to maintain functional HIV-specific CD8⁺ T cells, which are accounted for delayed HIV progression [54]. Nevertheless, blocking of TIM-3 signaling pathway could restore HIV-1-specific CD8⁺ T cell proliferation and cytotoxic capabilities [15,55].”

 

4. We have expanded section 2 by adding more information on immune checkpoint molecules and HIV, as suggested.

      Line 118-121: “HIV Nef protein drives the upregulation of PD-1 during in vitro infection, through its proline-rich motif and the activation of p38 signaling pathway [26]. PD-1 overexpression on CD8⁺ T cells is correlated with HIV viral load and disease progression [27].”

      Line 148-153: “HIV preferentially infects CTLA⁺ CD4⁺ T cells in vitro and negatively modulates CTLA-4 expression in the presence of Nef protein to allow productive viral replication [41]. A study using HAART-treated simian immunodeficiency virus (SIV)-infected rhesus macaques uncovers the memory CD4⁺ T cells expressing CTLA-4 contain high level of SIV DNA and infectious virus, suggesting CTLA-4 could be an additional target for eliminating viral reservoir [42].”

Line 161-167: “FoxP3⁺ CD8⁺ regulatory T (Treg) cell expressing high level of CTLA-4 is found to have no cytolytic potential and is directly correlated with high viremia in SIV-infected rhesus macaques [43]. A similar observation of increased CTLA-4⁺ FoxP3⁺ CD8⁺ Treg cells in untreated PLWH, and early initiation of HAART reduces this immunosuppressive population [44]. The same study also reports the elite controllers presenting a similar level of CTLA-4 on FoxP3⁺ CD8⁺ T cells as the HIV-negative individuals, which could be associated with the maintenance of T cell antiviral responses [44].”

Line 209: “It is also associated with the plasma HIV viral load and disease progression [57].” 

Line 220-221: “It is expected to similar in the case of HIV infection, yet limited evidence of LAG-3 blockade is shown.”

Line 231-233: “In consistent with the finding, a recent study demonstrates that the TIGIT⁺ natural-killer (NK) cells derived from PLWH lose the capability to produce TNF-a, IFN-γ and CD107a, which is positively associated with viral load [69].”

Line 237-240: “More recent study demonstrates that the combination blockade of LAG-3, CTLA-4, or TIGIT can increase the frequency of cells expressing CD107a and IL-2, which are associated with cytotoxicity and survival of HIV-specific CD4⁺ and CD8⁺ T cells in HAART-treated PLWH [72].”

 

5. We have added the HIV-oriented contents on sections 3.1, 3.2 and 3.3, as suggested.

Line 296-300: “Indeed, HIV-1 preferentially infected CD4⁺ T cells with high oxidative phosphorylation and glycolysis, as indicated by positive correlations between the metabolic activities in activated cells and HIV infection levels [88]. The activated, proliferating primary CD4⁺ T cells are highly susceptible to HIV-1 infection ex vivo for productive virus replication [89].”

Line 316-321: “Notably, higher frequency of central memory CD8⁺ T cells has been reported in untreated PLWH with low viral load, implying memory cells mediate effective viral control [95]. Central memory CD4⁺ and CD8⁺ T cells in PLWH express high levels of GLUT1 and mitochondrial mass [96]. Consistently, memory CD4⁺ T cells in vitro exhibit increased oxidative phosphorylation relative to aerobic glycolysis, which act as a major determinant of HIV infection [88,97].”

Line 357-367: “In chronic viral infection such as HCV, CD8⁺ T cell exhaustion is characterized by a general suppression of broad range of genes associated with metabolic functions; these include genes encoding mitochondrial and OXPHOS components [107]. Impaired glucose and mitochondrial metabolism, as indicated by mitochondrial membrane depolarization and markedly high level of ROS, are detected in exhausted HCV-specific T cells [107]. It is interesting to note that the distinct metabolic nature of exhaustion of CD8⁺ T cells in HIV infection is different from other viral diseases. In HIV context, glycolysis is the main energy source for CD8⁺ T cells in early, untreated PLWH and viral controllers, which is crucial for T cell killing activity [108]. Exhausted CD8⁺ T cells show reduction in glycolysis but increased OXPHOS capacity, and dysregulated mTOR pathway that could not be manipulated to reverse exhaustion in T cell [108].”

6. We agree with this and have incorporated your suggestion in these sections.

Line 405-411: “Upon entry to the CD4⁺ T cell, HIV-1 replication further causes an increase in glycolytic flux whereby virion production can take place efficiently, and eventually enhances virus-induced cell death in the infected cell [112]. Several key metabolites involved in glycolysis such as hexose-P, fructose 1,6-bisphosphate (FBP), glyceraldehyde-3P (G3P) and 3-phosphoglycerate (3PG) are evidently elevated by infected CD4⁺ T cells [113]. Moreover, suboptimal inhibition of glycolysis eliminates HIV-infected cells and impairs viral amplification in CD4⁺ T cells isolated from HAART-treated PLWH [88].”

Line 422-428: “The older generation of HAART treatment, in particular nucleoside reverse transcriptase inhibitors, triggers severe mitochondrial toxicity through inhibition of DNA polymerase gamma and followed by the depletion of mitochondrial DNA [117]. While the newer treatment with different classes of HAART drugs are able to improve metabolic fitness in CD8⁺ T cells, they are unable to restore mitochondrial impairment [118]. Robust functional mitochondria are coupled with a healthy bioenergetic phenotype and have a direct role in cellular exhaustion.”

Line 430-432: “Apart from that, early initiation of HAART allows rapid and close-to-complete normalization of T cell subsets, and preserves T cell functions [119], as well as prevents metabolic alterations that drives T cell exhaustion.”

Line 448-451: “Persistent immune activation and cytokines production are also detected in the transient controllers and strongly associated with the metabolites level [100], while opposite trends of the parameters are observed in the persistent controllers [121].”

Line 473-479: “By comparing the immunometabolic profiles of HIV-specific CD8⁺ T cells between elite controllers and non-controllers (HAART-treated individuals), a greater metabolic plasticity of CD8⁺ T cells is observed in the HIV controllers [126]. Further, while elite controllers possess HIV-specific CD8⁺ T cells that appear to be mitochondrial-dependent to fuel T cell responses in addition to glycolysis, HIV-specific CD8⁺ T cells from non-controllers show larger mitochondrial mass, which has been linked to dysfunctional mitochondria [126].”

7. Changes have been made throughout the section and a paragraph has been moved up to line 508-513.  Immune checkpoint inhibition clinical data are included in the last paragraph of each checkpoint molecules, in section 2.

Line 482-484: “Immune checkpoint inhibitors are often defined as effective immunotherapies in various cancer and chronic infections. Emerging evidences support that metabolic re-programming has direct impact on T cell differentiation and exhaustion [78,85,127].”

Line 490-493: “The effects of PD-1 on the metabolic functions and bioenergetics of activated CD4⁺ T cells have also been examined. Study has revealed ex vivo PD-1⁺ T cells could not uptake and utilize glucose, as shown by the inhibition of GLUT1 expression, glucose transport, and HK2 mRNA level in T cells upon receiving PD-1 signals [128].”

 

Line 497-499: “Nevertheless, there is still limited clinical data regarding the immune checkpoint anti-bodies and their impacts on T cell metabolic functions in HIV context.”

Line 506-507: “These findings emphasize the efficiency of PD-1 inhibition on restoration of metabolic fitness, and subsequently the T cell activation status.”

Line 513-515: “Treatment with dual checkpoint blockade serves a more potent strategy by targeting different pathways could enhance anti-viral responses and T cell metabolism.”

Line 516-517: “Considering the effect of HAART on immune checkpoint expression, studies have combined multiple therapies to investigate the T cell function and reversal of HIV latency.”

Line 522-525: “Therefore, combined treatment inclusive of immune checkpoint inhibitors remains a possible solution to achieve HIV remission.”

Line 540-546: “Intriguingly, metformin (also an anti-diabetic drug) reduces the frequency of PD1⁺ TIM3⁺ TIGIT⁺-expressing CD4⁺ T cells in HAART-treated PLWH [134]. Recent study demonstrates that CD4⁺ T cells from women PLWH with diabetes mellitus receiving metformin show lower glucose metabolic activity as compared to those without medication [135]. Thus, it is suggested that metformin could be an adjunct therapy to reduce metabolic activation, partially correct glucose metabolism in CD4⁺ T cell as well as reverse exhaustion [136]."

.”

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

In this manuscript titled “Immune Checkpoint Molecules and Glucose Metabolism in HIV-induced T Cell Exhaustion”, authors thoroughly introduced the roles of the immune checkpoint molecules and glucose metabolism in chronic infections and discussed how these two signals could be modulated to revive exhausted T cells in people infected with HIV. Overall, this is an interesting review. However, it lacks certain details, therefore, a major revision is needed.

 

Major issues:

 

1. Line 135-137, “CTLA-4 is upregulated in HIV–specific CD4+ T cells in untreated PLWH of viremic controllers, acute and chronic infections, excluding the elite controllers”, how about CD8 T cells? Actually, in the section of CTLA-4, CD8 T cells were rarely mentioned. Are there more studies about CTLA-4 on CD8 T cells in PLWH?

 

2. Line 194-197, “Studies demonstrate elevated glucose uptake, glycolysis and lactate secretion [63], accompanied by high expression level of glycolysis-associated proteins including glucose transporter 1 (GLUT-1) and hexokinase 2 (HK2) in activated T cells”, HK2 was mentioned multiple times in the review, but it was never introduced what HK2 is. In addition, HK2 didn’t appear in figure 1.

 

3. Line 252-255, “In accordance to this change, CD8+ T cell exhaustion, in chronic viral infection such as HCV, is characterized by a general suppression of broad range of genes associated with metabolic functions [80]. Study shows that the expression of glycolytic-associated genes is augmented in exhausted T cells compared to effector T cells during viral infection”, these two sentences contradict with each other. More details are needed here. For example, which genes are augmented in exhausted T cells compared to effector T cells during viral infection?

 

4. Line 325-327, “Enforcement of metabolic fitness at early phase of infection is important to counteract full scale cell exhaustion, and is therefore crucial for repressing disease progression”, this sentence is very vague, more details are needed.

 

5. Line 343-345, “Persistent immune activation and cytokines production are also detected in the transient controllers and strongly associated with the metabolites level”, this sentence is very confusing, how about in persistent controller subgroup?

 

6. Line 399-401, “Usage of immune checkpoint inhibitors coupled with metabolism enhancing drugs to restore T cell exhaustion serve as promising complementary therapeutic options for HIV”. The authors only introduced that HAART could improve metabolism, but didn’t introduce any metabolism enhancing drugs. If authors believe metabolism enhancing drugs are important to restore T cell exhaustion in HIV infections, then the authors need a separate section to introduce these drugs specifically for metabolism enhancement.

 

Author Response

We sincerely thank the reviewer for providing valuable feedback on our manuscript. We have incorporated the changes as suggested by the reviewer.

 

Major issues:

1. Line 161-167: More details on CD8 T cells in CTLA-4 section have been added in the manuscript, as follow: "FoxP3⁺ CD8⁺regulatory T (Treg) cell expressing high level of CTLA-4 is found to have no cytolytic potential and is directly correlated with high viremia in SIV-infected rhesus macaques [43]. A similar observation of increased CTLA-4⁺ FoxP3⁺ CD8⁺ Treg cells in untreated PLWH, and early initiation of HAART reduces this immunosuppressive population [44]. The same study also reports the elite controllers presenting a similar level of CTLA-4 on FoxP3⁺ CD8⁺ T cells as the HIV-negative individuals, which could be associated with the maintenance of T cell antiviral responses [44].”
2. Line 255-259: We have added a short description for HK2 in the text, as follow: “Studies demonstrate elevated glucose uptake, glycolysis and lactate secretion [79], accompanied by high expression level of glycolysis-associated proteins including glucose transporter 1 (GLUT1) and hexokinase 2 (HK2), a key enzyme engaged in glycolysis catalyzing the conversion of glucose to glucose-6-phosphate, in activated T cells [80].” However, we did not illustrate HK2 in the figure because HK2 is only one of the many enzymes in glycolysis.

                                                       

3. Line 357-367: We have removed the second sentence and revised this paragraph by adding in more details on the exhausted T cell metabolism. “In chronic viral infection such as HCV, CD8⁺ T cell exhaustion is characterized by a general suppression of broad range of genes associated with metabolic functions; these include genes encoding mitochondrial and OXPHOS components [107]. Impaired glucose and mitochondrial metabolism, as indicated by mitochondrial membrane depolarization and markedly high level of ROS, are detected in exhausted HCV-specific T cells [107]. It is interesting to note that the distinct metabolic nature of exhaustion of CD8⁺ T cells in HIV infection is different from other viral diseases. In HIV context, glycolysis is the main energy source for CD8⁺ T cells in early, untreated PLWH and viral controllers, which is crucial for T cell killing activity [108]. Exhausted CD8⁺ T cells show reduction in glycolysis but increased OXPHOS capacity, and dysregulated mTOR pathway that could not be manipulated to reverse exhaustion in T cell [108].“
4. We have added in more details, as suggested.

Line 426-429: “Robust functional mitochondria are coupled with a healthy bioenergetic phenotype and have a direct role in cellular exhaustion.”

Line 430-432 “Apart from that, early initiation of HAART allows rapid and close-to-complete normalization of T cell subsets, and preserves T cell functions [119], as well as prevents metabolic alterations that drives T cell exhaustion.”

5. We have revised this sentence and added more details about the persistent controllers.

Line 448-451: “Persistent immune activation and cytokines production are also detected in the transient controllers and strongly associated with the metabolites level [100], while opposite trends of the parameters are observed in the persistent controllers [121].”

Line 473-479: “By comparing the immunometabolic profiles of HIV-specific CD8⁺ T cells between elite controllers and non-controllers (HAART-treated individuals), a greater metabolic plasticity of CD8⁺ T cells is observed in the HIV controllers [126]. Further, while elite controllers possess HIV-specific CD8⁺ T cells that appear to be mitochondrial-dependent to fuel T cell responses in addition to glycolysis, HIV-specific CD8⁺ T cells from non-controllers show larger mitochondrial mass, which has been linked to dysfunctional mitochondria [126].”

6. Line 540-546: We have added a paragraph regarding the metabolic enhancing drug in section 5. “Intriguingly, metformin (also an anti-diabetic drug) could reduce the frequency of PD1⁺ TIM3⁺ TIGIT⁺ -expressing CD4⁺ T cells in HAART-treated PLWH [134]. Recent study demonstrated that CD4⁺ T cells from women PLWH with diabetes mellitus receiving metformin show lower glucose metabolic activity as compared to those do not receiving anti-diabetic medication [135]. Thus, it is suggested that metformin could be an adjunct therapy to reduce metabolic activation, partially correct glucose metabolism in CD4⁺ T cell as well as reverse exhaustion [136].”

Author Response File: Author Response.pdf

Reviewer 4 Report

Chan and colleagues distilled a complex topic into a very clear review. All sections are detailed yet easy to read and understand. I have only comment to improve the manuscript.

Line 52 - HIV-1 was defined before, use acronym 

Line 94 - PD-1 was defined before

Line 102 - possesses a motif

Line 105 - please clarify “they act as brake”

Line 349 - they targeted subunits of the mTOR complex

Author Response

We thank the reviewer for generous comments given to improve the manuscript. We have carefully addressed every comment and corrected them accordingly.

1. Line 51-54: We have replaced the “human immunodeficiency virus” with the acronym “HIV”.
2. However, the acronym PD-1 is only mentioned in the abstract. In the main text, PD-1 is first defined in line 94.
3. Line 104-107: The grammatical error has been corrected in the text.
4. Line 107-109: We have reclarified the phrase “they act as brake” as follow: “These negative regulatory motifs are important to the cell physiology as they transduce inhibitory signals that inactivate the effector T cells, which counteracts the TCR signaling.”
5. Line 455-458: The mistake has been rectified accordingly in the text. “mTOR complex subunits knockdown by CRIPSR interference or pharmacological inhibition, as well as inhibition of Enolase 1 in CD4+ T cells, prevent viral reactivation and suppress latency reversal by repressing Tat-dependent and Tat-independent transcription of HIV [107].”

Author Response File: Author Response.pdf

Reviewer 5 Report

Chan et al., have submitted a concise review entitled "Immune Checkpoint Molecules and Glucose Metabolism in HIV-induced T Cell Exhaustion". 

Overall, the authors made a substantial effort to clearly understand immune checkpoint inhibitions in HIV infection and T cell exhaustion. 

A great discussion is placed on T cell glucose metabolism regulation in immune checkpoint inhibitors in HIV infection. 

The discussion of PD-1, CTLA-4, LAG-3 and TIGIT are informative. 

 

This reviewer has some suggestions.

Why TIM-3 has not discussed much.

A table describing immune checkpoint inhibitors in HIV-induced T cell exhaustion and their current status in the clinical trials, followed by the mechanism of actions, would benefit this review. 

Author Response

We thank the reviewer for reviewing and providing insightful comments to improve our paper. We have carefully considered and addressed the comments accordingly.  

1. We have added the information on TIM3 to the section 2 in the manuscript, as follow. Line 179-202; 2.3. TIM-3:

"TIM-3 is initially identified as a Th1-specific transmembrane protein that characterizes the differentiated Th1 CD4⁺ T cell [49]. TIM-3 regulates T cell proliferation, production of pro-inflammatory cytokines IFN-g and peripheral tolerance [50], which are driven by the binding of galectin-9, a ligand of Tim-3 [51]. The TIM-3/galectin-9 pathway triggers inhibitory signaling, reduces IFN-g producing Th1 cells and induces cell death in activated Th1 cells [51].

During HIV infection, viral proteins such as Nef and Vpu exert opposing effects on TIM-3 expression level in infected CD4⁺ T cells. As shown by a recent in vitro study, HIV-1 Nef protein mediates the upregulation of TIM-3 through its dileucine motif and contradictorily, activates the infected cells through TCR signaling [52]. Conversely, Vpu protein downregulates TIM-3 surface expression on infected CD4⁺ T cells via its transmembrane domain and alters its subcellular localization [53]. These effects might because of the early expression of Nef to favor viral replication, while Vpu is expressed late to facilitate viral release.

In progressive HIV infection, high expression of TIM-3 on HIV-1-specific CD8⁺ T cell is correlated with the frequency of dysfunctional T cell population [15]. Regulatory T cell utilizes the TIM-3/galectin-9 pathway to suppress proliferation of HIV-specific CD8⁺ T cell; the protective allele HLA-B*27- and HLA-B*5-restricted CD8⁺ T cells present low level of TIM-3 upregulation can evade the regulatory T cell-mediated suppression [54]. This explains why HIV elite controllers possessing these HLA alleles are able to maintain functional HIV-specific CD8⁺ T cells, which are accounted for delayed HIV progression [54]. Nevertheless, blocking of TIM-3 signaling pathway could restore HIV-1-specific CD8⁺ T cell proliferation and cytotoxic capabilities [15,55].”

 

2. Thank you for the suggestion. While we agree that this is an important point, there is there is still limited information on this aspect related to T cell metabolism in HIV context. Furthermore, a recent review (Gubser, C., Chiu, C., Lewin, S. R., & Rasmussen, T. A. (2022). Immune checkpoint blockade in HIV. EBioMedicine, 76, 103840.) that focuses on the status of immune checkpoints in HIV, has also been published.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The manuscript is better now

Reviewer 3 Report

All my concerns resolved.