Hypoxia Enhances Immunosuppression by Inhibiting CD4+ Effector T Cell Function and Promoting Treg Activity

Abstract

Background/aims:
Hypoxia occurs in many pathological conditions, including inflammation and cancer. Within this context, hypoxia was shown to inhibit but also to promote T cell responses. Due to this controversial function, we aimed to explore whether an insufficient anti-tumour response during colitis-associated colon cancer could be ascribed to a hypoxic microenvironment.

Methods:
Colitis-associated colon cancer was induced in wildtype mice, and hypoxia as well as T cell immunity were analysed in the colonic tumour tissues. In addition, CD4+ effector T cells and regulatory T cells were cultured under normoxic and hypoxic conditions and examined regarding their phenotype and function.

Results:
We observed severe hypoxia in the colon of mice suffering from colitis-associated colon cancer that was accompanied by a reduced differentiation of CD4+ effector T cells and an enhanced number and suppressive activity of regulatory T cells. Complementary ex vivo and in vitro studies revealed that T cell stimulation under hypoxic conditions inhibited the differentiation, proliferation and IFN-γ production of TH1 cells and enhanced the suppressive capacity of regulatory T cells. Moreover, we identified an active role for HIF-1α in the modulation of CD4+ T cell functions under hypoxic conditions.

Conclusion:
Our data indicate that oxygen availability can function as a local modulator of CD4+ T cell responses and thus influences tumour immune surveillance in inflammation-associated colon cancer.

Full text

Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1271
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
Original Paper
Accepted: February 13, 2017
This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 Interna-
tional License (CC BY-NC-ND) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution
for commercial purposes as well as any distribution of modied material requires written permission.
DOI: 10.1159/000464429
Published online: March 08, 2017
© 2017 The Author(s)
Published by S. Karger AG, Basel
www.karger.com/cpb
© 2017 The Author(s)
Published by S. Karger AG, Basel
Infection Immunology, Institute of Medical Microbiology, University Hospital Essen,
Hufelandstr. 55, 45122 Essen, (Germany)
Tel. +492017231826, Fax +492017235602, E-Mail astrid.westendorf@uk-essen.de
Astrid M. Westendorf
Hypoxia Enhances Immunosuppression by
Inhibiting CD4+ Effector T Cell Function
and Promoting Treg Activity
Astrid M. Westendorfa Kathrin Skibbea Alexandra Adamczyka Jan Buera
Robert Geffersb Wiebke Hansena Eva Pastillea Verena Jendrossekc
aInstitute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen,
bGenome Analytics, Helmholtz Centre for Infection Research, Braunschweig, cInstitute of Cell Biology
(Cancer Research), University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Key Words
Colitis-associated colon cancer • Hypoxia • HIF-1a • T cell function
Abstract
Background/Aims: Hypoxia occurs in many pathological conditions, including inammation
and cancer. Within this context, hypoxia was shown to inhibit but also to promote T cell
responses. Due to this controversial function, we aimed to explore whether an insufcient
anti-tumour response during colitis-associated colon cancer could be ascribed to a hypoxic
microenvironment. Methods: Colitis-associated colon cancer was induced in wildtype mice,
and hypoxia as well as T cell immunity were analysed in the colonic tumour tissues. In addition,
CD4+ effector T cells and regulatory T cells were cultured under normoxic and hypoxic conditions
and examined regarding their phenotype and function. Results: We observed severe hypoxia
in the colon of mice suffering from colitis-associated colon cancer that was accompanied by
a reduced differentiation of CD4+ effector T cells and an enhanced number and suppressive
activity of regulatory T cells. Complementary ex vivo and in vitro studies revealed that T cell
stimulation under hypoxic conditions inhibited the differentiation, proliferation and IFN-γ
production of TH1 cells and enhanced the suppressive capacity of regulatory T cells. Moreover,
we identied an active role for HIF-1α in the modulation of CD4+ T cell functions under
hypoxic conditions. Conclusion: Our data indicate that oxygen availability can function as a
local modulator of CD4+ T cell responses and thus inuences tumour immune surveillance in
inammation-associated colon cancer.
Introduction
Colorectal cancer is a major cause of cancer-related death in many countries. In recent
years, new hypotheses on the mechanisms involved in colorectal carcinogenesis have been

in patients with ulcerative colitis, is strongly associated with the development of colon cancer
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Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1272
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb

driven by a prevalent activation of T helper cells [2]. If the immune response fails to resolve
the injury, a tumour-prone microenvironment with prolonged uncontrolled proliferation in
attempt to repair the damage may result. Epithelial oxygen tension plays a critical role in
  
described to subsist in a state of physiologically low oxygen level. More precisely, intestinal
epithelial cells have a steep oxygen gradient where the tips on the villi are hypoxic and
 
dramatic shift in tissue metabolism and perfusion, which results in heightening of hypoxia

elicits protective innate immune response, and activates an antimicrobial response by the
            
      
tumours and this has been attributed to its multiple contributions to chemoresistance,
radioresistance, angiogenesis, resistance to cell death, altered metabolism, and genomic
instability [4-7].
         
immune response. It is well established that T cells in the context of established progressing
tumours exhibit an anergic or exhausted state leading to T cell-intrinsic dysfunction [8].
Furthermore, a variety of solid tumours possess an increased amount of immunosuppressive
cells, such as regulatory T cells (Tregs) and immunosuppressive cytokines derived from the
Tregs or the tumour cells [9, 10]. Increasing evidence now suggests that oxygen availability
can regulate T cell differentiation and function, e.g. human T cells activated under hypoxia
are protected from activation-induced cell death [11]. Conversely, cultivating activated
  2       
apoptosis in certain T cell subsets [12]. Furthermore, it was demonstrated that in vitro

       
cancer, we combined both issues and examined whether the impaired colonic CD4+ T cell
response during colitis-associated colon cancer (CAC) could be associated with a hypoxic
microenvironment.
Materials and Methods
Mice
All animals used in this study were 8- to 12-week-old female and male mice bred and housed under
            
    
             

AOM/DSS protocol
           . Mice
 
   
Mice were

Histology and immunohistochemistry of colon tissues
            
            
    

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75.101.163.131 - 3/10/2017 3:06:16 AM

Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1273
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
by anti-pimonidazole sta     . In brief, tissue slides were


 
           
®.


 
    
           
   
            
 

Isolation of lamina propria lymphocytes from the colon
   , 


2

Clostridium histolyticum, 
min. The remaining tissue was separated from cells by passing the cell suspension through a 40 µm cell
strainer and washing with culture medium.

          

               
            
 


  
(both from BD Biosciences).
Proliferation assay
CD4+-            
+- T cells (4 × 10) were activated in vitro with plate-
2)
conditions or in a hypoxia chamber (Invivo2
2


Suppression assay
CD4++ Tregs were separated from the colons of healthy mice and mice with colitis-associated
              
CD4++ T-cell isolation kit II (Miltenyi Biotec,
+ responder T cells (1 × 10)
were either cultured alone or co-cultured with CD4+++) Tregs (1 × 10

).
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75.101.163.131 - 3/10/2017 3:06:16 AM

Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1274
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
To analyse the suppressive capacity of Tregs under hypoxia, CD4++ Tregs and CD4+ responder
           + responder T
cells (2 × 10) were either cultured alone or co-cultured with CD4++ Tregs (2×10  
            
     

TH1 differentiation assay
CD4+- 
sorter (BD Biosciences) and were activated in vitro
           
1-cell-polarization was measured

Transcriptome analyses
+
             

           
                

Technologies).
Quantitative RT-PCR
 

 

      
      
      

Statistics


of P 
Results and Discussion
Colitis-associated colon cancer induces a strong hypoxic and immunosuppressive
microenvironment
To assess whether hypoxia is induced during CAC and whether low oxygen levels may


   
           


To determine the presence of hypoxia within the colonic tumour tissues, the binding of
pimonidazole to macromolecules, which occurs in the absence of adequate oxygen levels,
was evaluated. Consistent with earlier reports, physiologic hypoxia was observed in healthy

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75.101.163.131 - 3/10/2017 3:06:16 AM

Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1275
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
in week 12 after the initial induction of colitis-associated colon cancer, an increased colonic
epithelial pimonidazole-uptake, extending to additional parts of the crypts, the submucosal
layer and the tumour tissue, respectively, was observed in mice suffering from colitis-
associated colon cancer (CAC, Fig. 1B).
+ T cells in the colon of healthy mice and mice
with CAC. In tumour-bearing colons, the proportion of CD4+

activity of CD4+ T cells by the determination of their cytokine production, we observed a
  +         
ex vivo in the colon of CAC mice compared to healthy controls (Fig. 2B). This is well in line
with the human situation, as decreased percentages of T1 cells were found in patients with
++
                
          + T cells. This implies
   

Besides the anergic or exhausted state of effector T cells in tumours, a variety of solid
tumours reveal an increase in immunosuppressive cells, such as Tregs [19, 20]. Importantly,
+ regulatory CD4+ T cells was detected in the colons of mice
suffering from CAC but not in the colon of healthy control mice (Fig. 2C). To further explore
the suppressive activity of Tregs in the hypoxic colonic tumour tissue, we sorted CD4++
Tregs from the colons of healthy and CAC-diseased mice and co-cultured them with CD4+
responder T cells. As demonstrated in Fig. 2D, we observed that the ability of CD4++
Tregs to suppress the proliferation of responder CD4+ T cells was enhanced when these
cells were isolated from CAC mice. This is well in line with recent studies demonstrating
that Tregs isolated from human and mouse colonic tumour tissues strongly suppressed the
+ and CD4+ T cells [21, 22].

      
           
             
Fig. 1. Colitis-associated colon cancer is associ-
ated with severe hypoxia in the colon. To induce
colitis-associated colon cancer, mice were given an
   
   -
    
images from the distal colon of healthy control mice
   
(B) To assess colonic hypoxia during colitis-associat-
ed colon cancer, mice were treated intraperitoneally
     -
tion of hypoxia in colonic mucosa was then per-
  
histological images from the distal colon of healthy

     

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75.101.163.131 - 3/10/2017 3:06:16 AM

Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1276
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
secretion was increased in the tumorous tissue (Fig. 2E). Collectively, we observed severe
hypoxia in the colon of mice suffering from CAC that was accompanied by a reduced CD4+
effector T cell differentiation and an enhanced number and suppressive activity of Tregs.
+ T cells
In order to analyse in more detail, whether hypoxia directly alters CD4+ T cell functions,
CD4+-

+- T cells stayed constant over the course of
Fig. 2. Colitis-associated colon cancer induces a strong immunosuppressive microenvironment in the colon.

 
+ T cells on gated viable cells are demonstrated.

on gated viable CD4+ T cells was determined. Absolute numbers of CD4++ per colon was calculated. (C)
+  
              
++
+ 
+ responder T cells with anti-

eFluor dye and inhibition was calculated. Inhibition of healthy control Tregs was set to 1 and the values of
-

-


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Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1277
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
the experiment, both under normoxic and hypoxic conditions. In contrast, the cell numbers

  + T cells was
            
Interestingly, from the second day onwards the proliferation of CD4+ T cells stimulated under
    + T cells stimulated under

indicate that hypoxia inhibits the proliferation of CD4+ T cells in vitro. These results are in
parts consistent with a former study, which demonstrates that the proliferation of human

Fig. 3.      
labelled CD4+- T cells were stimulated with

conditions. (A) Every day, the viable cell number
was determined by trypan blue staining. Bar dia-
grams represent the number of viable cells as mean
      
      

(B) At indicated time points, cells were analysed by
        -
liferation index was calculated by setting the mean
     
      -
responding stimulated cells accordingly. The recip-
rocal value is displayed as the proliferation index.
All experiments were performed in duplicates and
the mean value of three independent experiments
is shown. Bar diagrams represent the proliferation
  
-

Fig. 4.-
ed hypoxia-inducible genes in effector T
cells. CD4+- T cells were stimulated
    
or normoxic conditions for 4 h or 24 h re-
 
     
  
expression was normalized to the house-
      
the mean quantity of hypoxic versus nor-
moxic samples are displayed.
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75.101.163.131 - 3/10/2017 3:06:16 AM

Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1278
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
To get a comprehensive overview about the effector function of T cells under hypoxia,
transcriptome analyses of CD4+ effector T cells, stimulated under hypoxic or normoxic
        
          + T cells stimulated under
hypoxic conditions compared to CD4+ T cells stimulated under normoxic conditions (Table

9 cells have been reported
     
how hypoxia interferes with the generation of T9 cells.

        
            
         


           
conditions [28]. Consequently, we examined whether the impact of hypoxia on CD4+ effector
              

+ T cells stimulated under

  

        
            
slightly increased under hypoxia (Table 1).
 +- T cells were
stimulated under either normoxic or hypoxic conditions for 4 h and 24 h respectively, and

 

minimal changes in expression which is in line with the transcriptome analyses. Interestingly,


           
     + effector T cell function under
hypoxic conditions.
Table 1. Transcriptome analysis of selected genes in effector CD4+ T cells
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75.101.163.131 - 3/10/2017 3:06:16 AM

Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1279
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
Hypoxia inhibits the differentiation of TH1 cells
            
expression was altered in the colon of mice suffering from CAC, and under hypoxic conditions
in vitro          1 effector
+ T cells into T1 effector cells. For this, CD4+

      
Fig. 5.    
cells into T1 cells is impaired under
hypoxia. CD4+ T cells were stimulated
     
 1 differentiation ei-
ther under normoxic or hypoxic con-
ditions. After 4 days, the expression of

The histograms show the expression
+ among CD4+-
resentative experiment of three in-
dependent experiments is displayed.
    -
+ T cells and the corre-
sponding standard error of the mean.
Fig. 6.     
+ Tregs but in-
creases the inhibitory capacity. (A) To-
tal CD4+ T cells were stimulated with
   
or normoxic conditions for 4 h or 24
h. The cells were harvested and the
    ++ T

   + T cells and
    
    -
periment out of three independent
experiments is displayed. (B) CD4+
+ Tregs were co-cultured at a
  +
responder T cells in the presence or
     
-
sponder T cells was measured by the
loss of eFluor dye, and inhibition was
calculated. Inhibition of Tregs culti-
vated in normoxia was set to 1 and the
values of Tregs cultivated in hypoxia
were adjusted to them. Bar diagrams
             -

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75.101.163.131 - 3/10/2017 3:06:16 AM

Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1280
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
       1 cells
          
1


Hypoxia modulates the function of regulatory T cells
         

+ T cell under normoxic and hypoxic conditions and analysed

    
               
demonstrating that hypoxia does not enhance the Treg abundance in vitro [27].
To determine whether hypoxia alters the function of Tregs, transcriptome analyses of
CD4++ Tregs, stimulated under normoxic and hypoxic conditions, were performed in
accordance to the analyses of CD4+ effector T cells. As already detected for CD4+ effector T
   

to normoxic conditions (Table 2). This is not surprising as Tregs are not considered as
          
hypoxia may elevate the inhibitory activity of Tregs, we isolated CD4++ Tregs from
the spleen of mice and co-cultured them with CD4+ responder T cells under normoxic and
  

         
    


+ effector T cells, and therefore could be involved in the reduced
differentiation of CD4+
on CD4+- and CD4++ T cell in the colon of healthy control mice and mice suffering
from CAC. Furthermore, we performed transcriptome analysis regarding the expression of
+- and CD4++ T cell cultured under hypoxic and normoxic conditions.
+- T cells in the
colon of mice suffering from CAC compared to healthy control mice as well as after stimulation
under hypoxia in vitro
on CD4++ Tregs during CAC and when exposed to hypoxia in vitro (Fig. 7A, B), which
has been described to enhance the suppressive capacity of Tregs through the interaction
Table 2. Transcriptome analysis of selected genes in CD4+ Tregs
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Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1281
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
Fig. 7.-

   -

-
sion of CD4+- effector T cells and CD4++ Tregs stimulated in vitro under hypoxic and normoxic
conditions.
Fig. 8.
hypoxia-inducible genes in regulatory
T cells. Total CD4++ T cells were
    -
ther hypoxic or normoxic conditions for 4
       
     
     
expression was normalized to the house-
      
the mean quantity of hypoxic versus nor-
moxic samples are displayed.

immunosuppression rather than the induction of effector T cell exhaustion.

   
   
              

mediated colitis [41].
Taken together, our data indicate that oxygen availability can function as a local modulator

in the cancerous crypts that was accompanied by a reduced CD4+ effector T cell function and
in vitro experiments show
           
that also other factors than hypoxia are involved in the inhibition of an effective anti-tumour
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Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1282
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
response in vivo. Further studies are needed to unravel whether pharmacologic strategies
           
associated colon cancer.
Acknowledgements

       

Disclosure Statement

References
 
 
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 
 
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 
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 
promotes hepatocellular carcinoma metastasis by way of up-regulating hypoxia-inducible factor 1alpha
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 
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 
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
 
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 

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Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1283
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
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

 
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 
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 
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 
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 
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75.101.163.131 - 3/10/2017 3:06:16 AM

Cell Physiol Biochem 2017;41:1271-1284
DOI: 10.1159/000464429
Published online: March 08, 2017 1284
Westendorf et al.: Hypoxia Impairs T Cell Function
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry © 2017 The Author(s). Published by S. Karger AG, Basel
www.karger.com/cpb
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
 


 
upregulated on regulatory T cells during chronic virus infection enhances the suppression of CD8+ T cell


 



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