Peer review of: The CD153 vaccine is a senotherapeutic option for preventing the accumulation of (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
In this manuscript, the authors present what I believe is the first evidence illustrating the 
development of a vaccine against senescent cells. Specifically, they target senescent T cells that 
accumulate in visceral adipose tissue during obesity. These senescent cells are defined by the 
surface markers CD4+ CD44high CD62Llow PD-1+ CD153+. Vaccination was with a CD153 
peptide conjugated to KLH and resulted in increased anti-CD153 antibody production in mice. The 
authors found the antibody lowered senescent T cells in adipose tissue of mice fed a high fat diet 
for 10 weeks. This was accompanied by improved glucose tolerance and insulin sensitivity. They 
provide in vitro evidence (complement-dependent cytotoxicity assay) to support the hypothesis 
that the CD153 antibodies evoked have the capacity to remove CD153+ cells. In vivo, they show 
fewer crown-like structures around adipocytes, a marker of white adipose tissue inflammation. 
 
The experiments are carefully executed, and numerous controls are used, including using KLH 
alone to immunize mice, using different adjuvants, and comparing endogenous anti-CD153 to a 
commercially available antibody. 
 
Overall, this is an intriguing proof-of-concept study. Applications are limited in that CD153+ cells 
do not increase significantly in visceral fat with normal aging (Fig. 1D) and CD153+ T cells are 
important for preventing lung infection (Nat Microbio 2018). Thus, the utility of a CD153-CpG 
vaccine as a senolytic therapy has both safety and utility issues. The last sentence of the 
discussion might be tempered accordingly. 
 
In addition, in Figure 4, panels C & D are expressed as percentage of crown-like structures around 
adipocytes and CD153+CLS. The data should be expressed as actual values. N = 3 mice per 
group. How many sections? How many fields counted? How many actual CLS observed? And how 
many stained positive for CD153? This is important to provide an indication as to what extent 
senescent T cells must be cleared to have a physiological impact. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In the manuscript entitled “The CD153 vaccine with CpG adjuvant is a novel serotherapeutic option 
for removing senescence-associated T cells from visceral adipose tissues “authors describe 
development of the vaccine to target CD153 expressing, senescent associated (SA)- T 
lymphocytes. Infiltration of immune cells in fat is a hallmark of obesity related insulin resistance. It 
has been demonstrated previously that among other immune cell types CD153+PD-
1+CD44hiCD4+ T cells accumulate in the adipose tissue with obesity and contribute to obesity 
induced insulin resistance. Attempt to clear or prevent accumulation of SA-T lymphocytes in fat 
could be an attractive approach to inhibit inflammation and improve metabolic dysfunction 
associated with obesity. 
However, the manuscript has major issues which need to be addressed before it is considered for 
publication. 
Mice vaccinated with CD-153-CpG vaccine described in the paper have decrease food consumption 
and body weight compared to mock vaccinated and high fat feed controls. Decrease food 
consumption in mice indicates stress or illness, unless intervention impacts CNS. As authors stated 
CD-153-CpG vaccinated mice after 11 weeks of high fed feeding have improved insulin glucose 
tolerance and insulin sensitivity and less CD153+SA T lymphocytes in the visceral fat. Many study 
endpoints described in the experiment with high fat feeding are sensitive to food intake, body 
weight and the level of adipocity, and it is important to clarify whether reported outcomes 
involving these measures are truly reflective of efficacy of vaccine or are a result of an adverse 
event/ toxicity of the immunization. 

To clarify these authors should perform “pair feeding”, and have additional control groups in the 
experiment. They need to vaccinate chow fed mice and look if there is any adverse or off target 
effect. 
Vaccination against CD153 positive lymphocytes might be risky and has very limited if any path to 
translation. CD153 expressing CD4+ lymphocytes play an important role in modulating immune 
response and fight infection. Sallin MA, Kauffman KD, Riou C, Du Bruyn E, Foreman TW, Sakai S, 
Hoft SG, Myers TG, Gardina PJ, Sher A, Moore R, Wilder-Kofie T, Moore IN, Sette A, Lindestam 
Arlehamn CS, Wilkinson RJ, Barber DL. Host resistance to pulmonary Mycobacterium tuberculosis 
infection requires CD153 expression. Nature Microbiology2018 2018/11/01;3(11):1198-205. 
Vaccine which has prolonged sustainable effect against this ligand might have serious adverse 
effects on health. Author’s should quote this paper and address this issue in the limitation of the 
approach. 
There are publications showing beneficial effects of senescent cell clearance on metabolic health. 
Authors should quote these publications to make the paper less superficial 
Xu M, Palmer AK, Ding H, Weivoda MM, Pirtskhalava T, White TA, Sepe A, Johnson KO, Stout MB, 
Giorgadze N, Jensen MD, LeBrasseur NK, Tchkonia T, Kirkland JL. Targeting senescent cells 
enhances adipogenesis and metabolic function in old age. Elife2015 Dec 19;4:e12997. 
Schafer MJ, White TA, Evans G, Tonne JM, Verzosa GC, Stout MB, Mazula DL, Palmer AK, Baker DJ, 
Jensen MD, Torbenson MS, Miller JD, Ikeda Y, Tchkonia T, van Deursen JM, Kirkland JL, LeBrasseur 
NK. Exercise Prevents Diet-Induced Cellular Senescence in Adipose Tissue. Diabetes2016 
Jun;65(6):1606-15. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
In the manuscript “The CD153 vaccine with CpG adjuvant is a novel serotherapeutic option for 
removing senescence-associated T cells from visceral adipose tissues”, the authors show that 
vaccination can delete cells that contribute to a senescent phenotype and improve obesity-induced 
metabolic disorders. I was asked to comment on the immunological aspects of this manuscript. 
The vaccination protocol and assessment of the response to vaccination are all appropriate and 
standard for the field. Quantification of antibody production and function (in a cytotoxic assay) are 
all done properly as is the flow cytometric analysis of cell populations. The only issue that I have is 
with the animals used in these studies. In the TLR7 ligand administration study, female mice were 
used, while in the HFD loading study, male mice were used. It is well known that males and 
females have different immune responses to vaccination. I would have liked to see both sexes be 
used for both experiments. Aside from this point, this is a well done, nicely presented study. 
 
 
 
Reviewer #4: 
Remarks to the Author: 
Summary: The authors developed a vaccine with a peptide from CD153 and CpG 1585. The 
vaccine boosted anti-CD153 antibodies, in particular several IgG subclasses, for a prolonged 
period. The authors them demonstrated that the CD153-CpG vaccine was able to reduce 
senescence-associated T cells (SA-T cells), which express CD153, in visceral adipose tissue of 
obese mice, most likely mediated by IgG2 antibodies against CD153. 
 
Overall Critique: This work is important and the data are convincing that the vaccine has potential 
as a therapy to deplete SA-T cells. The approach to develop the vaccine is not particularly novel, 
although the application is novel. I only have a few concerns. 
 
Concerns: 
1. Why did the authors chose CpG 1585 (Invivogen), a class A CpG, rather than a Class B or C 
CpG? It would be interesting to compare each of these classes of TLR9 agonists in your model. 

2. Is the mouse CD153#D peptide homologous in humans? In other words, could this approach be 
easily translated to vaccine development for humans? 
3. Can you provide statistical analyses for Figure 1A and Figure 1C? 
4. Why only 3 mice in Figure 1F?