Peer review of: T cell exhaustion and a failure in antigen presentation drive resistance to the graft-versus- (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

Zhou et al report on graft-versus-leukemia studies in a model system that relies on T cells that 
recognize the minor histocompatibility antigen H60. The authors show that Leukemia-derived H60 
was inefficiently cross-presented and they use different approaches to enhance GVL effects 
including an agonistic CD40 antibody, blockade of PD1, Tim-3, TIGIT or LAG3. T cell exhaustion 
and leukemia burden are used a major read-outs. The main finding is that FGK45 administered at 
the time of transplant was that the agonistic CD40 antibody was most potent at enhancing GVL 
effects. 

Major comments: 
1. H60 is an artificial antigen, in patients multiple hematopoietic antigens (including major MHC 
molecules such as HLA-DP1 which are not matched) are recognized by the donor T cells after allo-
HSCT. The authors should reproduce their findings in a different leukemia model that relies at least 
on one additional antigen. This would make their finding relevant for the clinic. 
2. FGK45 alone augmented the anti-H60 response, even in the absence of BC-CML, - this shows 
that there were sources of H60 other than the leukemia derived H60. How can the authors be sure 
that the TetH60+ (H60 reactive) T cells are then really leukemia reactive? 
3. The authors state that the anti CD40 antibody was most effective - why did they not perform a 
survival study of the leukemia bearing mice? 
4. The authors use a CML model (Figure 1) - CML is hardly anymore an indication for allo-HSZT. 
The studies should focus on AML. 
5. Figure 6: anti PD1 treatment after allo-HSCT causes GVHD -did the authors analyze GVHD 
target organs? 
6. The H60-expressing mouse that relies on bone marrow (BM) transduced with retroviruses that 
express cDNAs encoding the human bcr-abl and NUP98/HOXA9 translocations is artificial. Genetic 
models of AML are more relevant for the clinic. 
7. The authors show that a-CD40 at time of transplantation augments GVL -this is very early 
treatment - in the clinic relapse is treated when it shows MRD positivity or hematological relapse. 
Such scenario should be tested by the authors (established AML treated with a-CD40). 

Reviewer #2 (Remarks to the Author): 

In this manuscript, Zhou et al. examined mechanisms of the failure in graft-versus-leukemia (GVL) 
using a mouse model wherein GVL is mediated by minor histocompatibility antigen H60-specific T 
cells that are trackable with MHC tetramer. In this model, H60 specific memory CD8 T cells 
generated after vaccination were adoptively transferred into irradiated recipient mice along with T 
cell-depleted bone marrow as well as H60 expressing leukemia cells. They found that H60-specific 
memory CD8 T cells failed to control the expansion of the leukemia cell population due to 
inefficient H60 antigen presentation. Instead, H60-antigen presented by leukemia cells contributed 
to the differentiation of these memory CD8 T cells into exhausted T cells. Such exhausted CD8 T 
cells included a PD-1+ Tcf1+ subset, which was previously observed during chronic viral infection 
and in solid tumors. PD-1 blockade diminished T cell exhaustion and improved GVL. Furthermore, 
the authors showed that treatment with anti-CD40 antibody augmented H60-specific memory CD8 
T cell-mediated GVL. Similar to what was seen in the model of memory CD8 T cell transfer, naïve 
CD8 T cells differentiated into exhausted T cells after adoptive transfer. 

Overall, this work provides a comprehensive analysis of how CD8 T cells respond to leukemia cells 
and also shows the mechanisms of the failure in GVL as well as a potential therapeutic approach to 
improve GVL. This manuscript could be improved if the authors address the following issues and 
questions. 

1. In the mouse model used in this study, the authors irradiated H60-expressing B6 mice and then 
donor bone marrow, H60-specific memory CD8 T cells, and H60+ blast crisis chronic myelogenous 
leukemia (BC-CML) were adoptively transferred into these irradiated mice. Why were H60-
expressing B6 mice used as a recipient instead of H60 negative regular B6 mice? This information 
will be helpful for readers to understand the experiments in this manuscript. 

2. In Fig. 1C, D, and E, a burden of BC-CML is shown in the presence of H60-specific CD8 T cells. 
Do these H60-specific CD8 T cells contribute to the control of BC-CML? Does the expansion of the 
BC-CML population further increase if H60-specific CD8 T cells are not transferred? 

3. Experimental designs of supplemental Fig. 2D and E are not clear. In these experiments, bone 
marrow cells were transplanted into irradiated B6.H60 mice but the designs give the impression 
that bone marrow cells were isolated from B6.H60 mice. Please revise them. 

4. On pages 12 and 20, the authors mentioned that they were unable to detect H60-specific CD8 T 
cells that were PD1+ CXCR5+, a subset shown to respond to PD-1-blockade in LCMV infection. 
What clone and fluorochrome were used for staining of CXCR5 (there is no information in the MM 
section)? CXCR5 expression in CD8 T cells is dim compared with B cells, and specific clone(s) as 
well as brightest fluorochrome are required to detect CXCR5 on exhausted CD8 T cells. 
Alternatively, the existence of CXCR5 can be transcriptionally confirmed. Do the authors have the 
data of CXCR5 mRNA levels in PD1 positive Tim3 low H60-specific CD8 T cells (compared with 
those in PD1 positive Tim3 high H60-specific CD8 T cells or naïve CD8 T cells)? Since expression 
patterns of other markers (Tcf-1, CD39, KLRG1, and Ly108) are very similar to the LCMV system, 
the Reviewer is wondering if CXCR5 staining would fail due to a technical issue. If the authors do 
not have conclusive data about CXCR5 expression, the Reviewer suggests removing these parts 
from the manuscript. 

5. It is very difficult to distinguish the lines in the histograms in Fig. 7D. Please revise them. 

6. On page 13 line 11, the authors mentioned: “FGK45 primarily acted on host cells as there was 
no reduction in GVL in recipients of CD40-/- BM (Fig. 7F).” What are the host cells? Do host cells 
mean recipient cells? Please clarify them. 

7. It is very difficult to understand the rationale, the experimental design, and the data of Fig. 7G. 
Why were mixed bone marrow chimera mice made? Why were these mixed bone marrow chimera 
mice re-irradiated for transplantation? This part needs more information for a better understanding 
of the data. 

8. On page 16 line 15, (Tn) should be right after “CD8 T cells from either unmanipulated C3H.SW 
donors”. 

9. In Fig. 10A and B, it will be better to show Tox expression levels in unstimulated naïve CD8 T 
cells as a negative control. 

10. On page 16 the last line to page 17 the first line, the authors said “TOX expression in TetH60-
negative cells in Tn-recipients was bimodal, with discrete populations of PD-1 High TOX High and 
PD-1 low TOX low cells (Fig. 10E). FGK45 increased the frequency of TetH60 negative PD-1 high 
TOX high cells in spleen, perhaps by enhancing alloreactive CD8 cell expansion.” However, Fig. 
10E analyzes Tcf-1 expression (no TOX expression data). So, the Reviewer thinks that Tcf1 
expression in TetH60-negative cells in Tn-recipients was bimodal. Also, Fig. 10E shows that PD-1 
high cells are Tcf-1 low. Conversely, PD-1 low cells are Tcf-1 high. Please revise and clarify this 
part.