Article

LDHA-Associated Lactic Acid Production Blunts

Tumor Immunosurveillance by T and NK Cells
Graphical Abstract Authors
Almut Brand, Katrin Singer,
Gudrun E. Koehl, ...,
Wolfgang Mueller-Klieser,
Kathrin Renner, Marina Kreutz

Correspondence
marina.kreutz@ukr.de

In Brief
Brand et al. link altered tumor glucose
metabolism and immune escape and
show that increased lactic acid
production by LDHA in cancer cells
impairs cytokine production, in particular
IFN-g, in tumor-infiltrating T cells and NK
cells, thereby inhibiting tumor
immunosurveillance and promoting
tumor growth.

Highlights
d Human melanoma metastases exhibit a ‘‘Warburg
phenotype’’ with high lactic acid levels

d LDHA-associated lactic acid production and acidification

lead to immune evasion

d Lactic acid and acidification diminish NFAT levels and T and

NK cell activation

d LDHA expression in melanoma patients correlates with

survival and T cell activity

Brand et al., 2016, Cell Metabolism 24, 657–671

November 8, 2016 ª 2016 Elsevier Inc.
http://dx.doi.org/10.1016/j.cmet.2016.08.011
Cell Metabolism

Article

LDHA-Associated Lactic Acid Production

Blunts Tumor Immunosurveillance by T and NK Cells
Almut Brand,1 Katrin Singer,1 Gudrun E. Koehl,2 Marlene Kolitzus,1 Gabriele Schoenhammer,1 Annette Thiel,1
Carina Matos,1 Christina Bruss,1 Sebastian Klobuch,1 Katrin Peter,1,3 Michael Kastenberger,1 Christian Bogdan,4
Ulrike Schleicher,4 Andreas Mackensen,5 Evelyn Ullrich,5,6 Stefan Fichtner-Feigl,2,3 Rebecca Kesselring,2
Matthias Mack,3,7 Uwe Ritter,8 Maximilian Schmid,1,8 Christian Blank,9 Katja Dettmer,10 Peter J. Oefner,10
Petra Hoffmann,1,3 Stefan Walenta,11 Edward K. Geissler,2 Jacques Pouyssegur,12,13 Andreas Villunger,14,15
André Steven,16 Barbara Seliger,16 Stephan Schreml,17 Sebastian Haferkamp,17 Elisabeth Kohl,17 Sigrid Karrer,17
Mark Berneburg,17 Wolfgang Herr,1 Wolfgang Mueller-Klieser,11 Kathrin Renner,1,3 and Marina Kreutz1,3,18,*
1Department of Internal Medicine III, University Hospital Regensburg, 93053 Regensburg, Germany
2Department of Surgery, University Hospital Regensburg, 93053 Regensburg, Germany
3Regensburg Center for Interventional Immunology, University of Regensburg, 93053 Regensburg, Germany
4Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen,

Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg, 91054 Erlangen, Germany

5Department of Internal Medicine 5, Hematology/Oncology, University Hospital Erlangen, 91054 Erlangen, Germany
6Cellular Immunology, Pediatric Stem Cell Transplantation and Immunology, Department for Children and Adolescents Medicine of the

University Hospital Frankfurt, Goethe-University, 60590 Frankfurt, Germany

7Department of Internal Medicine II - Nephrology, University Hospital Regensburg, 93053 Regensburg, Germany
8Institute of Immunology, University of Regensburg, 93053 Regensburg, Germany
9Division of Immunology, the Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Amsterdam 1066CX, the Netherlands
10Institute of Functional Genomics, University of Regensburg, 93053 Regensburg, Germany
11Institute of Pathophysiology, University Medical Center of the Johannes Gutenberg University Mainz, 55128 Mainz, Germany
12Institute of Research on Cancer and Aging, University of Nice-Sophia Antipolis, Centre A. Lacassagne, 06189 Nice, France
13Centre Scientifique de Monaco (CSM), 98000 Monaco, Monaco
14Medical University Innsbruck, Biocenter, Division of Developmental Immunology, 6020 Innsbruck, Austria
15Tyrolean Cancer Research Institute, 6020 Innsbruck, Austria
16Martin Luther University Halle-Wittenberg, Institute of Medical Immunology Halle/Saale, 06112 Halle, Germany
17Department of Dermatology, University Hospital Regensburg, 93053 Regensburg, Germany
18Lead Contact

*Correspondence: marina.kreutz@ukr.de
http://dx.doi.org/10.1016/j.cmet.2016.08.011

SUMMARY INTRODUCTION

Elevated lactate dehydrogenase A (LDHA) expres-
sion is associated with poor outcome in tumor pa-
tients. Here we show that LDHA-associated lactic
acid accumulation in melanomas inhibits tumor sur-
veillance by T and NK cells. In immunocompetent
C57BL/6 mice, tumors with reduced lactic acid
production (Ldhalow) developed significantly slower
than control tumors and showed increased infiltra-
tion with IFN-g-producing T and NK cells. However,
in Rag2–/–gc–/– mice, lacking lymphocytes and NK
cells, and in Ifng–/– mice, Ldhalow and control cells
formed tumors at similar rates. Pathophysiological
concentrations of lactic acid prevented upregulation
of nuclear factor of activated T cells (NFAT) in T and
NK cells, resulting in diminished IFN-g production.
Database analyses revealed negative correlations
between LDHA expression and T cell activation
markers in human melanoma patients. Our results
demonstrate that lactic acid is a potent inhibitor of
function and survival of T and NK cells leading to
tumor immune escape.
Accelerated glucose metabolism in tumor cells, the so-called
‘‘Warburg effect,’’ is based on the upregulation of glucose trans-
porter 1 (GLUT1) and glycolytic enzymes such as lactate dehy-
drogenase A (LDHA), which is essential for the conversion of
pyruvate into lactate. Glycolysis requires continuous export of
lactate from cells by monocarboxylate transporters (MCTs),
which co-transport lactate and protons. As a result, lactate
and protons (‘‘lactic acid’’) accumulate in the tumor environment.
In primary human tumors, high levels of lactate correlate with
incidence of distant metastases (Walenta et al., 2000), and the
level of LDHA correlates with the size and clinical stage of renal
cell carcinomas and gastric cancer (Girgis et al., 2014; Sun et al.,
2014). Based on these data, increased glycolysis by tumor cells
appears to promote their growth, progression, and metastasis.
The relationship among increased lactate production, tumor
growth, and metastasis has been investigated in different mouse
models (Fantin et al., 2006; Rizwan et al., 2013; Xie et al., 2014).
Attenuation or disruption of Ldha resulted in reduced tumor
growth, and the authors attributed the diminished tumorigenicity
to the compromised ability of tumor cells to grow under hypoxia
or the importance of LDHA for tumor-initiating cells, respectively.
Tumor-derived lactic acid inhibits the differentiation and acti-
vation of monocytes and T cells in vitro (Dietl et al., 2010; Fischer

Cell Metabolism 24, 657–671, November 8, 2016 ª 2016 Elsevier Inc. 657
 (legend on next page)
658 Cell Metabolism 24, 657–671, November 8, 2016
et al., 2007; Puig-Kröger et al., 2003). The tumor-promoting ef-
fect of lactic acid might therefore be, in part, related to its immu-
nosuppressive effects. Concordant with this hypothesis, Shime
et al. demonstrated that lactic acid regulates expression and
secretion of the tumor-promoting cytokine interleukin 23 (IL-23)
(Shime et al., 2008). Furthermore, Husain et al. reported reduced
numbers of myeloid-derived suppressor cells (MDSCs) in the
spleens of mice carrying Ldha-depleted tumors (Husain et al.,
2013). More recently, Colegio et al. showed that lactic acid
promotes development of M2-like macrophages by inducing
hypoxia-inducible factor 1a (Colegio et al., 2014). These results
clearly indicate the importance of glycolysis and tumor-derived
lactic acid not only for the tumor growth itself, but also for the
immune cell balance in the tumor environment.
Here we studied how lactic acid-induced changes modify the
anti-tumor immune response. We describe an important inter-
play between tumor-derived lactic acid and immune cells in
the tumor environment.
RESULTS
Human and Mouse Melanoma Tumors Exhibit the
Warburg Phenotype
Bogunovic et al. described an immune expression signature
as a predictor of survival in a cohort of patients with meta-
static melanoma (Bogunovic et al., 2009). Using this dataset,
we found a negative correlation between LDHA expres-
sion and survival in these patients (Figure 1A). Furthermore,
high LDHA expression correlated with low CD3d expression
(r value = 0.396, p value = 7.9 3 10 3), indicating a negative
impact of lactic acid on T cell infiltration. To clarify whether
LDHA expression is associated with increased lactate levels,
bioluminescence analyses of melanoma of different stages
and adjacent healthy skin were performed. Cutaneous metas-
tases exhibited dramatically elevated lactate levels compared
to primary melanomas and healthy skin (Figure 1B). Further-
more, glucose levels were significantly reduced in invasive pri-
mary melanomas and metastases. Interestingly, high lactate
and low glucose levels overlapped in a minority (n = 4) of
patients (Figure 1C and Table S1).
We hypothesized that melanoma is a good model to study
the relation between tumor glucose metabolism and immune
cell infiltration. Small hairpin RNAs (shRNAs) complementary
to Ldha were used to reduce expression of Ldha in B16.SIY mu-
rine melanoma cells (Ldhalow cells). Untransfected cells or cells
transfected with a nonspecific, scrambled shRNA were used
as controls. Levels of Ldha mRNA and LDHA protein were
reduced in Ldhalow1 and Ldhalow2 clones, compared to controls
(Figure 1D). Since the relative abundance of LDHA versus lactate
dehydrogenase B (LDHB) determines the enzymatic activity of
the tetrameric LDH enzyme complex, we also analyzed LDHB
expression. LDHB was significantly upregulated at the mRNA,
but not the protein, level in Ldhalow clones in vitro (Figure 1E).
In contrast, all clones expressed similar levels of Glut1 mRNA
(Figure 1F).
To measure the enzymatic activity of LDH, we analyzed lactate
in cell supernatants. As expected, Ldhalow tumor clones
secreted significantly less lactate than control cells in vitro (Fig-
ure 1G). To determine whether the clones exhibited a stable
phenotype in vivo, 1 3 105 tumor cells were injected subcutane-
ously into immunocompetent C57BL/6 mice. Established tumors
were dissected and analyzed. Levels of Ldha mRNA, but not
Glut1 mRNA were significantly lower in Ldhalow tumors than
in controls; Ldhb mRNA expression was diminished only in
Ldhalow2 tumors (Figures 1H–1J). We used quantitative biolumi-
nescence imaging to determine the distribution of glucose and
lactate in frozen sections of isolated tumors; glucose and lactate
levels were lower in Ldhalow tumors (data not shown and Fig-
ures 1K and 1L). When we re-cultured tumor cells from Ldhalow
and control tumors grown in C57BL/6 mice, we observed that
lactate levels remained significantly different between the cell
types (data not shown).
Next, we investigated whether the attenuation of glycolysis
would also affect other metabolic pathways that are involved
in tumor growth control and immune evasion by measuring
expression of indoleamine 2,3-dioxygenase 1 and 2 (Ido1,
Ido2), cyclooxygenase 1 and 2 (Cox1, Cox2), Arg1, and Nos2.
In tumor lysates we found no significant differences in the
expression of these genes between Ldhalow and control tumors
(Figures S1A–S1F). Thus, knockdown of Ldha resulted in a stable

Figure 1. The Warburg Phenotype Is Present in Human and Mouse Melanoma Tumors
(A) Overall survival of 44 metastatic melanoma patients with high LDHA (blue) and low LDHA (red) expression levels, calculated with the ‘‘R2: Tumor Melanoma
Metastatic – Bhardwaj – 44 – fRMA – u133p2’’ dataset (http://r2.amc.nl).
(B and C) Quantification of tissue levels of lactate (B) and glucose (C) in human biopsies of melanoma in situ (Mis), primary melanoma (M), cutaneous metastases
of melanoma (M Met), and healthy tissue of the respective patients (Ctrl) by induced bioluminescence imaging. Each dot represents one biopsy (32 patients were
analyzed), and horizontal lines indicate the mean (one-way ANOVA, Tukey’s post test).
(D) qRT-PCR analysis (left panel) of Ldha mRNA in untransfected B16.SIY cells (WT), cells transfected with a scrambled shRNA sequence (Ctrl), and cells
transfected with shRNAs against Ldha (Ldhalow1 and Ldhalow2). The results are presented relative to the level of 18S rRNA. Each dot represents an individual
experiment, and horizontal lines indicate the mean. Immunoblot analysis (right panel) of LDHA and phospho-LDHA (p-LDHA) in whole-cell extracts. Actin was
used as loading control. One representative experiment out of three is shown.
(E) The left panel shows qRT-PCR analysis of Ldhb mRNA, and the right panel shows protein levels of LDHB, analyzed as described in (D).
(F) qRT-PCR analysis of Glut1 mRNA, analyzed as described in (D).
(G) Levels of lactate in the supernatants of cells, cultured for 24 hr (n R 21 independent experiments, mean and SEM). Dashed line represents lactate level in
culture medium.
(H–J) qRT-PCR analysis of Ldha (H), Ldhb (I), and Glut1 mRNA (J) in tumors of C57BL/6 mice after subcutaneous injection of 105 control (Ctrl), Ldhalow1, or
Ldhalow2 cells. The results are presented relative to the level of 18S rRNA. Each dot represents an individual mouse, and horizontal lines indicate the mean.
(K) Induced metabolic bioluminescence imaging of intra-tumor levels of lactate in sections of tumors grown from control or Ldhalow cells. Two representative
images are shown.
(L) Quantification of intra-tumor lactate levels identified as in (K) (n = 3–5 mice per group, mean and SEM, multiple tumor areas per sample analyzed). Statistical
analyses were performed using unpaired Student’s t test for (D–J and L).

Cell Metabolism 24, 657–671, November 8, 2016 659

Figure 2. Growth of B16 Ldhalow and Panc-Ldhanull Tumors Is Controlled in C57BL/6 Mice
(A) Proliferation of control (Ctrl), Ldhalow1, and Ldhalow2 cells, based on 3H-thymidine incorporation, after 24 hr (n R 7 independent experiments, mean
and SEM).
(legend continued on next page)
660 Cell Metabolism 24, 657–671, November 8, 2016
tumor cell phenotype with no effects on the other metabolic
pathways analyzed.
T Cells and NK Cells Control Tumor Growth in Low
Lactate Tumors
Little is known about the effects of glycolytic restriction on anti-
tumor immunity. We therefore studied the growth of Ldhalow and
control B16.SIY melanoma cells in vitro and in vivo.
All clones, with the exception of Ldhalow1, proliferated similarly
in vitro (Figure 2A), and no differences were noted in cell-cycle
distribution (Figure S2A). Proliferation of Ldhalow cells could be
related to their increased mitochondrial content and activity
(Figures S2B and S2C). Metabolic analyses with 13C-labeled
glucose revealed that Ldha silencing increased the flux into
tricarboxylic acid cycle metabolites (Figure S2D). In line, oxygen
consumption related to ATP production was elevated in Ldhalow
clones and proliferation was severely diminished by oligomycin,
indicating that proliferation of ‘‘low’’ lactate but not ‘‘high’’ lactate
clones depends on oxidative phosphorylation (OXPHOS) (Fig-
ures S2E and S2F). Interestingly, both clones seem to depend
on glutamine, as proliferation decreased by glutamine depriva-
tion (Figure S2F).
Following injection of 1 3 106 Ldhalow or control tumor cells
into C57BL/6 mice, tumors grew at similar rates (Figure 2B). In
support of our hypothesis, that lower numbers of tumor cells
allow tumor growth to be controlled, 90% of mice injected with
1 3 104 Ldhalow cells were tumor free on day 18, whereas 70%
of mice injected with control cells developed tumors until this
time point (Figure 2C). To allow analysis of tumor immune cell
infiltration, mice were injected with 1 3 105 tumor cells. Tumors
originating from Ldhalow cells grew more slowly than those from
control cells, indicating that tumor control is still possible with
this tumor load. No sex-specific differences in tumor growth
were observed (Figure 2D).
To investigate whether lymphocytes and/or NK cells affect the
growth of tumors, we injected 1 3 105 tumor cells into immuno-
deficient Rag2–/– mice (lacking B and T cells) and Rag2–/–gc–/–
mice (lacking B, T, and NK cells). Growth of control tumors
was similar in immunocompetent and immunodeficient mice,
indicating that neither T cells nor NK cells are capable to restrict
growth of tumors with high lactate secretion (Figure 2E). In
contrast, in Rag2–/– mice, Ldhalow tumors showed accelerated
growth (Figure 2F). Lack of lymphocytes and NK cells in
Rag2–/–gc–/– mice completely abolished the growth difference
between Ldhalow and control tumors (Figures 2E and 2F).
Together, these results show that growth control by T and NK
cells is possible in Ldhalow but not control B16.SIY tumors.
Next, we extended our analysis to another tumor system. Ldha
knockout in the pancreatic adenocarcinoma cell line Panc02-H7
resulted in no detectable LDHA protein expression (Figure 2G),
strongly diminished lactate production (Figure 2H), and similar
proliferation in vitro (Figure 2I). In contrast, tumor development
in C57BL/6 mice was markedly delayed and reduced when in-
jecting Ldha knockout cells (Panc-Ldhanull) compared to con-
trols (Figure 2J). In vitro analyses revealed a strong dependence
of Panc-Ldhanull cells on OXPHOS (Figures S2G and S2H).
Increased Numbers of Cytotoxic Effector Cells in
Ldhalow Compared to Control Tumors
Flow cytometry was used to analyze the immune cells present in
tumors grown from 1 3 105 Ldhalow or control cells, isolated at
different time points after cells were injected into mice (see Fig-
ure 3A for gating strategy). Using the pan-leukocyte marker
CD45, we found that approximately 5% of the cells analyzed
were hematopoietic cells. We did not observe a relationship
between tumor size and the number of infiltrating leukocytes
(Figure 3B). However, Ldhalow tumors grown in in C57BL/6 (Fig-
ure 3C), but not in Rag2–/– mice (Figure 3D), had a greater propor-
tion of CD45+ immune cells than control tumors.
In addition, the composition of the immune cell infiltrate and
the percentage of myeloid cells, NK cells, and T cells relative
to levels of total CD45+ cells were determined. Myeloid cells
(CD11b+) and myeloid suppressor cells (MDSCs, CD11b+Gr-1+)
were present in high numbers in all tumors; the percentage of
myeloid cells did not correlate with tumor size when tumors up
to 1,000 mm3 in size were analyzed (Figure 3E). In Rag2–/– and
C57BL/6 mice, a higher frequency of CD11b+ myeloid cells
was detected in control tumors than in Ldhalow tumors, indi-
cating that lactate production promotes myeloid cell infiltration,
regardless of the presence of T cells or the strain of mice (Figures
3F and 3G). A greater proportion of MDSCs was only detected in
control tumors from Rag2–/– mice (Figures 3H–3J).
Myeloid cells accumulate in progressing tumors in humans
and mice and limit the anti-tumor immune responses of effector
cells such as NK and T cells. We therefore investigated the
numbers of NK1.1+ and CD3+ cells in the tumor types. Again,

(B) Growth of tumors after subcutaneous injection of 106 control (Ctrl) or Ldhalow cells into C57BL/6 mice. Data represent mean tumor volume ± SEM (n = 4–5 mice
per group).
(C) Tumor-free survival of C57BL/6 mice after subcutaneous injection of 104 wild-type (WT), control (Ctrl), Ldhalow1, or Ldhalow2 cells (n = 10 mice per group).
(D) Tumor growth after subcutaneous injection of 105 cells into male (m) and female (f) C57BL/6 mice as described in (B) (n = 4–5 mice per group). In statistical
analyses, growth of tumors was compared between control cells and Ldhalow cells among mice of the same sex.
(E and F) Tumor growth after subcutaneous injection of 105 control (Ctrl) cells (E) or Ldhalow cells (F) into C57BL/6, immunodeficient Rag2–/– mice, and Rag2–/–gc–/–
mice (n = 8–10 mice per group ± SEM). In statistical analyses, growth of tumors in C57BL/6 mice was compared to Rag2–/– mice and Rag2–/–gc–/–, respectively
(black asterisks), and growth of Ldhalow tumors in Rag2–/– mice was compared with growth of Ldhalow tumors in Rag2–/–gc–/– mice (red asterisks).
(G) Immunoblot analysis of LDHA and LDHB in whole-cell extracts of untransfected Panc02-H7 cells (WT) and clones after transfection with Ldha CRISPR/Cas9
plasmids (Panc-Ldhahigh, Panc-Ldhanull). Actin was used as loading control. One representative experiment out of three is shown.
(H) Lactate level in the supernatants of untransfected WT and clones (Panc-Ldhahigh, Panc-Ldhanull) cultured for 24 hr (n R 6 independent experiments, mean and
SEM, unpaired Student’s t test). Dashed line represents lactate level in culture medium.
(I) Proliferation of Panc cells measured as described in (A) (n R 5 independent experiments).
(J) Growth of tumors after subcutaneous injection of 105 Panc tumor cells described in (G) in C57BL/6 mice. Data represent mean tumor volume ± SEM, n = 10
mice per group. Growth of WT tumors was compared to Panc-Ldhahigh tumors (gray asterisks); WT and Panc-Ldhahigh were compared to Panc-Ldhanull (black
asterisks) using unpaired Student’s t test.

Cell Metabolism 24, 657–671, November 8, 2016 661

 Figure 3. B16 Ldhalow and Panc-Ldhanull Tumors Contain High
Numbers of Anti-tumor Effector Cells
(A) Gating strategy for flow cytometry analysis of leucocytes (CD45+),
living singular cells (DAPI- FSC-Wlow), myeloid cells (CD11b+), NK cells
(NK1.1+), MDSCs (CD11b+Gr-1+), T cells (CD3ε+), CD4+ T cells
(CD3ε+CD4+), and CD8+ T cells (CD3ε+CD8a+) in tumors from C57BL/6
mice after subcutaneous injection of 105 control (Ctrl) or Ldhalow cells.
Numbers in graphs indicate the percentage of cells. Plots of data from
one representative Ldhalow tumor are shown.
(B–Q) Correlations of CD45+ leukocytes among the entire cell pop-
ulations derived from B16 Ctrl and Ldhalow tumors of C57BL/6 mice with
tumor volume (B) and correlation of myeloid cells (E), MDSCs (H), NK
cells (K), and T cells (N) among living CD45+ leucocytes in tumors of
C57BL/6 mice with tumor volume. Each symbol represents an individual
mouse; correlations were calculated using a Pearson test. Percentage of
CD45+ leukocytes among the entire cell populations derived from tu-
mors of C57BL/6 mice (C) and percentage of immune cell populations
within the CD45+ cell population: myeloid cells (F), MDSCs (I), NK cells
(L), T cells (O), CD8+ T cells (P), and CD4+ T cells (Q). Percentage of
CD45+ leukocytes among the entire cell populations derived from tu-
mors of Rag2–/– mice (D) and percentage of immune cell populations
among the CD45+ cell population: myeloid cells (G), MDSCs (J), and
NK cells (M) (all identified as in A).
(R and S) Percentage of T cells (R) and NK cells (S) among CD45+ cells in
tumors of C57BL/6 mice after subcutaneous injection of 105 Panc-
Ldhahigh and Panc-Ldhanull cells. Each symbol represents an individual
mouse; small horizontal lines indicate the mean (unpaired Student’s
t test).

662 Cell Metabolism 24, 657–671, November 8, 2016

Figure 4. Ldhalow Tumors Contain Higher Amounts of Functionally Active CD8+ T Cells and NK Cells
(A and B) qRT-PCR analysis of Ifng mRNA in control (Ctrl) and Ldhalow tumors grown in C57BL/6 mice (A) and Rag2–/– mice (B). The results are presented relative
to the level of 18S rRNA. Each symbol represents an individual mouse; small horizontal lines indicate the mean.
(C–F) Proportions of IFN-g+ cells among CD8+ T cells (C) and NK cells (E), and granzyme B+ cells among CD8+ T cells (D) and NK cells (F) in tumors from C57BL/6
mice, measured by flow cytometry. Each symbol represents an individual mouse; small horizontal lines indicate the mean (unpaired Student’s t test).

no relation between NK cell infiltration and tumor size was found
(Figure 3K). However, the number of NK cells was increased in
Ldhalow tumors grown in C57BL/6 and Rag2–/– mice, compared
with control tumors (Figures 3L and 3M). Furthermore, we
observed no correlation between T cell infiltration and tumor
size, but the number of T cells was increased in Ldhalow tumors
grown in C57BL/6 mice (Figures 3N and 3O). The frequency of
CD3+CD8+, but not CD3+CD4+, T cells was significantly greater
in Ldhalow tumors than in control tumors (Figures 3P and 3Q). In
Panc-Ldhanull tumors derived from C57BL/6 mice, we also
observed an increase in CD3+ T cells by trend compared to
Panc-Ldhahigh tumors (Figure 3R). Furthermore NK1.1+ cells
were significantly enriched, whereas no change was detected
regarding myeloid cells (Figure 3S and data not shown).
Our findings suggest a link between tumor lactate levels and
the local composition of tumor-infiltrating immune cells. Since
tumors expressing high levels of Ldha modulate immune cell
infiltration and differentiation in the spleens of tumor-bearing
mice (Husain et al., 2013), we analyzed myeloid and lymphoid
cells in the blood and spleen of tumor-bearing C57BL/6 mice.
More myeloid (suppressor) cells and NK cells were found in
the blood of all tumor-bearing mice than in the blood of healthy
mice, whereas the numbers of CD3+ T cells were significantly
reduced in the blood of tumor-bearing mice (Figures S3A–
S3D). A similar trend was observed for immune cells in the
spleen (Figures S3E–S3H). We detected no significant differ-
ence in the composition of immune cells in the blood and spleen
of mice bearing Ldhalow versus control tumors, indicating that
lactate has a local effect on immune cells in the tumor
environment.
Lactic Acid Impairs Cytokine Production of Tumor-
Infiltrating T Cells and NK Cells
As T cell abundance alone is not informative regarding the anti-
tumor potency of T cells, we investigated the functional compe-
tence of tumor-infiltrating T cells. qRT-PCR analysis revealed
significantly higher mean levels of interferon-g (Ifng) mRNA in ly-
sates from Ldhalow tumors as compared to lysates from control
tumors grown in C57BL/6 mice (Figure 4A); a similar trend
was observed in immunodeficient Rag2–/– mice (Figure 4B). In
contrast, there was no significant difference in the levels of
mRNAs of tumor necrosis factor (Tnf) and vascular endothelial
growth factor a (Vegfa) in Ldhalow versus control tumors in
C57BL/6 mice (Figures S4A and S4B). Ldhalow and control tu-
mors were isolated from C57BL/6 mice, and levels of IFN-g
and granzyme B were measured by flow cytometry in T cells
and in NK cells. We found that the proportions of IFN-g and
granzyme B producing CD8+ T cells were significantly higher in
Ldhalow tumors than in control tumors (Figures 4C and 4D),
whereas no significant differences in the numbers of IFN-
g+CD4+ and IL-17+CD4+ T cells were observed between tumor
types (Figures S4C and S4D). NK cells also showed an increased
expression of IFN-g and granzyme B in Ldhalow tumors
compared to control tumors (Figures 4E and 4F). Thus, Ldhalow

Cell Metabolism 24, 657–671, November 8, 2016 663

Figure 5. Lactic Acid Suppresses Cytokine Production in CD8+ T Cells and NK Cells and Promotes Apoptosis
(A) Flow cytometric analysis of IFN-g+ and IL-2+ cells among stimulated CD8+ T cells (upper panel) and stimulated NK cells (lower panel) after incubation in the
absence (–) or presence (LA) of lactic acid, acidification (pH 6.4), or sodium lactate (NaL) for 24 hr. One representative experiment of three is shown.
(B) qRT-PCR analysis of Ifng mRNA expression in unstimulated or stimulated (PMA/Ionomycin) CD8+ T cells in the absence (–) or presence (LA) of lactic acid after
3 hr. Two independent experiments are shown. The results are presented relative to the level of 18S rRNA.
(C) Quantification of Annexin V+7-AAD+ CD8+ T cells isolated from spleens of C57BL/6 mice (WT) or vav-Bcl-2 mice after incubation in the absence (–) or presence
(LA) of lactic acid, acidification (pH), and sodium lactate (NaL) for 24 hr (n = 3 independent experiments, mean and SEM, paired Student’s t test).
(D and E) Percentage IFN-g+ T cells (upper panel) and IL-2+ T cells (lower panel) after coculture with either wild-type (WT), control (Ctrl), or Ldhalow cells without (D)
or with (E) IFN-g pre-treatment (n = 3–4 independent experiments, mean and SEM, unpaired Student’s t test).

tumors contain a higher proportion of anti-tumor effector cells
than control tumors, which might impede their growth.
Lactic Acid, but Not Its Sodium Salt, Inhibits Function
and Survival of T Cells and NK Cells
Knockdown of Ldha in mouse melanoma cells significantly
reduced the level of lactate in tumors grown from these cells (Fig-
ures 1K and 1L), and lactic acid has been shown to affect expres-
sion of IFN-g, IL-2, and granzyme B in cultured human T cells
(Fischer et al., 2007). We investigated whether lactic acid also in-
hibits expression of cytokines in mouse T and NK cells in vitro.
We stimulated mouse CD8+ T cells and NK cells in the absence
or presence of lactic acid, sodium lactate, or acidification corre-
sponding to 15 mM lactic acid (pH 6.4) and measured intracel-
lular IFN-g and IL-2 by flow cytometry. Lactic acid reduced the
expression of IFN-g on protein and mRNA level (Figures 5A
and 5B). NK cells were more vulnerable to the effects of lactic
acid, as 15 mM lactic acid already completely blocked IFN-g
production (Figure 5A). Acidification alone also diminished cyto-
kine production, but to a lesser extent compared to 15 mM lactic
acid. In contrast, sodium lactate had no effect (Figure 5A). Higher
levels of lactic acid (20 mM) or the respective acidification (pH
5.8) induced apoptosis of T cells irrespective of the expression
level of Bcl-2 (Figure 5C). Similar results were obtained with
NK cells (Figure S5A). Lactic acid did not modulate the expres-
sion of activation or exhaustion markers on activated CD8+ 2C
T cells but impaired upregulation of CD25 when present during
the activation process of naive CD8+ T cells (Figure S5B).

664 Cell Metabolism 24, 657–671, November 8, 2016

Figure 6. Lactic Acid Uptake by CD8+ T Cells Leads to Intracellular Acidification and Inhibition of NFAT Upregulation upon Stimulation
(A) Lactate uptake in CD8+ T cells after incubation with 15 mM 13C1 lactate and HCl corresponding to the pH of 15 mM lactic acid with or without an MCT1 inhibitor
for 4 hr (one experiment performed in duplicates).
(B) Intracellular pH of activated CD8+ T cells after incubation with lactic acid (0–20 mM LA) for 30 min using pH-controlled buffers (pH 4.5, pH 5.5, pH 6.5, and
pH 7.5) for calibration. One representative experiment of three is shown.
(C and D) Intracellular ATP levels of unstimulated and stimulated (PMA/Ionomycin) CD8+ T cells (C) and NK cells (D) in the absence (–) or presence of lactic acid
(LA). One experiment in triplicates (C) and two independent experiments (D) were performed.
(E and F) NFATc1 expression in unstimulated and stimulated (PMA/Ionomycin) CD8+ T cells (E) and NK cells (F) in the absence (–) or presence (LA) of 15 mM lactic
acid, HCl (pH 6.4), and sodium lactate (NaL) for 24 hr by flow cytometry (n R 3 independent experiments for all treatments, except for HCL n = 2). Data calculated
relative to control (PMA/Ionomycin). Data represent mean and SEM; paired Student’s t test.

We also measured expression of IFN-g and IL-2 in SIY-
specific CD8+ 2C T cells co-cultured with Ldhalow and control
B16.SIY tumor cells. CD8+ 2C T cells cultured with control tumor
cells expressed lower levels of IFN-g and IL-2 (Figure 5D).
In some experiments, tumor cells were pre-incubated with
IFN-g to upregulate MHC class I expression, which allows better
recognition by SIY-specific T cells. We measured higher
levels of cytokines in these cultures, and there was still a
detectable difference between Ldhalow and control co-cultures
(Figure 5E).
These data demonstrate that high levels of lactic acid prevent
expression of IFN-g by mouse CD8+ T and NK cells. Secretion of
lactate and protons by melanoma cells seems to reduce the pro-
portions of effector T and NK cells (Figures 3P and 3L), and their
production of anti-tumor cytokines such as IFN-g (Figures 5A
and 5B), which likely promotes tumor immune evasion and
growth.
Intracellular Acidification Caused by Lactic Acid
Disturbs NFAT Expression
Based on our findings with human T cells (Fischer et al., 2007),
we hypothesized that uptake of lactic acid by CD8+ T cells leads
to intracellular acidification. Therefore, mouse CD8+ T cells were
incubated with 13C1-labeled sodium lactate in the presence of
HCl to mimic ‘‘lactic acid’’ conditions and measured intracellular
13
C1-lactate levels. Comparable to human T cells, mouse T cells
showed an uptake of 13C1-lactate, which was poorly inhibited by
pre-incubation with a monocarboxylate transporter 1 (MCT1) in-
hibitor (Figure 6A). The MCT1 inhibitor did not prevent the inhibi-
tion of IFN-g expression or apoptosis induction (data not shown).
Uptake of 13C1-lactate was accompanied by an intracellular
accumulation of unlabeled lactate, which indicates that high
extracellular lactate levels disturb efflux of lactate from CD8+
T cells (data not shown). As lactate is transported together with
protons, we measured intracellular acidification. Exposure of

Cell Metabolism 24, 657–671, November 8, 2016 665

 (legend on next page)

666 Cell Metabolism 24, 657–671, November 8, 2016

CD8+ T cells to lactic acid dose-dependently decreased the
intracellular pH (Figure 6B). Intracellular acidification was paral-
leled by a drop in ATP levels in T and NK cells, indicating an
impaired energy metabolism (Figures 6C and 6D).
We then analyzed signaling pathways and transcription fac-
tors involved in IFN-g transcription such as nuclear factor of
activated T cells (NFAT) and NF-kB proteins (Sica et al., 1997).
Besides its activation by calcineurin, NFAT is upregulated during
activation of T cells on mRNA and protein level (Yan et al., 2013).
In T and NK cells, exposure to lactic acid or HCl inhibited upre-
gulation of NFAT during activation (Figures 6E and 6F), whereas
IkB degradation, a prerequisite for NF-kB activation, was not
altered (Figure S6A). Lactic acid also caused a slight decrease
in phosphorylated AKT and P38 MAP kinase (Figures S6B and
S6C). These data suggest that inhibition of NFAT is mainly
responsible for the suppression of IFNg expression in T and
NK cells.
IFN-g Is Required for Immune Control of Ldhalow Tumors
Since Ldhalow tumors were infiltrated by greater proportions of
IFN-g+ CD8+ T cells and NK cells than control tumors (Figures
4C and 4E), we speculated that the growth of Ldhalow tumors is
regulated via IFN-g production of T and/or NK cells. In this
case, absence of IFN-g expression by immune cells would miti-
gate the growth difference between Ldhalow and control tumors.
We injected 1 3 105 Ldhalow and control tumor cells into C57BL/6
mice and C57BL/6 mice with a disrupted Ifng gene (Ifng–/– mice)
and compared the growth of tumors from our cell lines.
Again, the growth of tumors from Ldhalow versus control cells
significantly differed in C57BL/6 mice. This difference was
completely lost in Ifng–/– mice. In fact, Ldhalow tumors grew
more rapidly in Ifng–/– mice than in C57BL/6 mice, whereas
growth of control tumors was not changed (Figure 7A). These
findings indicate the potential role of IFN-g in immunosurveil-
lance and growth control of Ldhalow tumors.
IFN-g is a pleiotropic cytokine that has different effects on host
and tumor cells. It directly inhibits tumor cell proliferation but also
modulates stroma cell function and activation. To distinguish be-
tween the direct effects of IFN-g on tumor cells and its effects on
the tumor stroma, we injected 1 3 105 Ldhalow or control tumor
cells, which express the IFN-g receptor 1 (IFNGR1) into Ifngr1–/–
mice. Under these conditions, IFN-g secretion by mouse stroma
cells could only act directly on the tumor cells that were injected,
but not on host cells. Just as in Ifng–/– mice, the growth rates of
Ldhalow and control tumors were similar in Ifngr1–/– mice. This
indicates that IFN-g regulates the growth of tumors indirectly,
by acting on cells in the tumor environment (Figure 7B).
Granzyme B expression in tumor-infiltrating CD8+ T cells was
reduced in highly glycolytic tumors (Figure 4D); granzyme B and
perforin are required for the cytotoxic activity of T and NK cells.
Therefore, 1 3 105 Ldhalow and control tumor cells were injected
into mice that lack perforin (Pfp–/– mice), and tumor growth was
monitored. Unexpectedly, and in contrast to Ifng–/– mice, the
growth of Ldhalow and control tumors differed significantly in
Pfp–/– mice (Figure 7C). This finding indicates that reduced
growth of Ldhalow tumors in mice does not result from a per-
forin-dependent cytotoxic activity of T cells. Accordingly, we
did not detect lysis of Ldhalow or control tumor cells by mouse
CD8+ T cells in vitro (data not shown). Based on these results,
we concluded that the reduced growth of Ldhalow tumors could
in part be related to the cytotoxic effects of NK cells, because
IFN-g has been shown to be important for NK cell activation
and suppression of tumor growth (Takeda et al., 2001).
The percentages of NK cells in tumors grown in Ifng–/– and
Ifngr1–/– mice were much smaller than those of C57BL/6 mice
(Figure 7D), whereas in spleens of tumor-bearing Ifng–/– mice
NK cell numbers were diminished in control, but not in Ldhalow
tumors, compared to healthy C57BL/6 mice (Figure S7A). In
contrast, the number of myeloid (suppressor) cells did not differ
significantly between Ldhalow tumors in C57BL/6, Ifng–/–, and
Ifngr1–/– mice (data not shown). However, a trend for enrichment
of myeloid (suppressor) cells was observed in spleens of Ifng–/–
mice compared to healthy C57BL/6 mice (Figures S7B and
S7C). Higher levels of CD8+ T cells were detected in Ldhalow tu-
mors of Ifng–/– or Ifngr1–/– mice compared to C57BL/6 mice, but
the percentage of activated, CD25+CD8+ T cells was signifi-
cantly higher in Ldhalow tumors grown in C57BL/6 mice,
compared to knockout mice (Figures 7E and 7F). IFN-g therefore
appears to be important for T cell activation and NK cell recruit-
ment in mice.
To correlate our findings with patient data, we analyzed im-
mune infiltration of cutaneous melanoma metastases, which
exhibit high lactate levels (Figure 1B) in comparison to adjacent
healthy tissue. Both tissues were heavily infiltrated by T cells;
however, only healthy adjacent tissue exhibited activated
CD8+CD25+ T cells, indicating that lactate levels may contribute
to immunosuppression in human melanoma as well (Figure 7G).
In line, database analyses (TCGA Research Network: http://
cancergenome.nih.gov/) revealed a negative correlation be-
tween LDHA expression and T cell activation markers such as
granzyme K (GZMK) and CD25 in human melanoma (Figures
7H and 7I).
We conclude that activation of tumor-infiltrating immune cells
is determined by LDHA expression and lactic acid levels in the

Figure 7. Correlation between LDHA, IFN-g, and CD25 Expression in Mouse and Human Melanoma
(A) Growth of control (Ctrl) and Ldhalow tumors in C57BL/6 mice and Ifng–/– mice (mean tumor volume ± SEM, n = 5 mice per group). Growth of control and Ldhalow
tumors was compared in C57BL/6 mice (black asterisks); growth of Ldhalow tumors was compared with growth of Ldhalow tumors in knockout mice (red
asterisks).
(B) Tumor growth in C57BL/6 mice and Ifngr1–/– mice as described in (A).
(C) Tumor growth in Pfp–/– mice as described in (A) (n = 7–8 mice per group).
(D–F) Quantification of NK cells (D) and CD8+ T cells (E) among living leucocytes, and CD25+ cells among CD8+ T cells (F) in control (Ctrl) and Ldhalow tumors
(identified as in Figure 3A). Each symbol represents an individual mouse (small horizontal lines indicate the mean, unpaired Student’s t test).
(G) Flow cytometric analyses of CD8+ cells among CD3+ T cells (upper panel) and CD25+ cells among CD8+ T cells (lower panel) from biopsies of human
cutaneous melanoma metastases and adjacent healthy tissue (control).
(H and I) Correlations of LDHA with GZMK (H) or CD25 (I) gene expression determined in a dataset containing 470 melanoma tumors (‘‘R2: Tumor Skin Cutaneous
Melanoma – TCGA – 470 – rsem – tcgars’’). Patient samples were ordered by LDHA levels (red squares). Pearson’s correlation was calculated.

Cell Metabolism 24, 657–671, November 8, 2016 667

tumor environment. High levels of lactate and concomitant
acidification limit immune cell activation and allow immune
escape.
DISCUSSION
A positive correlation between LDHA, high lactate levels, and tu-
mor progression has been documented in various tumor entities
including melanoma (Girgis et al., 2014; Walenta et al., 2000).
Immunohistochemical analysis revealed high expression of the
tetrameric LDHA (LDH-5) in human melanoma and metastases,
which was associated with reduced disease-free and overall sur-
vival of patients (Zhuang et al., 2010). In contrast, high levels of
tumor-infiltrating T cells predict a favorable prognosis in patients
with melanoma and other types of cancer (Bogunovic et al.,
2009; Galon et al., 2006). A new aspect in this context is the link-
age between immune cell activation, tumor cell LDHA expres-
sion, and lactate levels in the tumor environment. Our study
shows that high LDHA expression is associated not only with
poor prognosis, but also with lower expression of T cell markers
(Bogunovic et al., 2009). Analyses of melanoma biopsies further
revealed that cutaneous melanoma metastases exhibit high
levels of lactate that could contribute to the immunosuppression
in melanoma patients. Lactate levels in primary melanomas were
comparable to the adjacent tissue, indicating that other mecha-
nisms lead to immunosuppression in these patients. In this re-
gard, studies by Chang et al. and Ho et al. have shown that
accelerated glucose metabolism of tumor cells leads to glucose
deprivation and suppresses anti-tumor T cell effector function
(Chang et al., 2015; Ho et al., 2015). Accordingly, we detected
lower glucose levels in 50% of primary melanoma biopsies.
Therefore, highly glycolytic tumors can use different strategies
to suppress T cell activation: glucose restriction and/or lactate
accumulation.
As in vivo data on the effects of lactic acid are lacking, we
studied its impact in mouse tumor models and observed that
the level of lactic acid affects immune cell populations and
thereby regulates tumor growth.
In our melanoma model we found that tumor-derived lactic
acid reduced the numbers and activity of CD8+ T cells and NK
cells in vitro and in vivo. Both immune cell populations were
important for the immune control of tumors with low lactate
secretion as tumor growth was accelerated in immunodeficient
Rag2–/– mice and Rag2–/–gc–/– mice. Ablation of IFN-g also
blurred the growth difference between Ldhalow and control
tumors. In these mice, T cells seemed to be non-activated, as
they lack CD25 expression, and NK cells were almost undetect-
able. These findings reveal the importance of IFN-g for T and NK
cell homeostasis in the tumor environment. In line, IFN-g plays an
important role in the development of hepatic NK cells (Wu et al.,
2012). In addition, IFN-g can limit tumor growth by inhibiting
angiogenesis (Qin et al., 2003) or directly suppressing tumor
cell proliferation (Kakuta et al., 2002). VEGF-regulated angiogen-
esis did not appear to be affected by lactic acid in our model as
similar levels of Vegf mRNA were detected in Ldhalow and control
tumors. Nor did it seem that IFN-g was directly inhibiting prolifer-
ation as the difference in growth of Ldhalow versus control tumors
observed in C57BL/6 mice was not seen in Ifngr1–/– mice in
which tumor cells still express IFNGR1.
668 Cell Metabolism 24, 657–671, November 8, 2016
How does lactic acid impair NK and T cell activation and func-
tion? Activated T cells perform glycolysis and require efficient
secretion of lactate and protons, as intracellular accumulation
of lactate disturbs their metabolism (Fischer et al., 2007). The
export of lactate by MCTs depends on a gradient from cyto-
plasmic to extracellular lactate concentrations. Accordingly,
we show that high concentrations of extracellular lactic acid
lead to lactate and proton uptake and subsequently lowered
the intracellular pH in T cells. MCT1 inhibition could not block
acidification, as other MCTs or transporter-independent diffu-
sion may be involved (Parks et al., 2013). In contrast, Pilon-
Thomas and colleagues described that acidification did not
affect cytoplasmic pH in T cells (Pilon-Thomas et al., 2016). Dif-
ferences in the kinetics could explain these conflicting results.
Lactate uptake and accumulation disturbed energy metabolism
as ATP levels were lowered. In line with these data, sodium
lactate or lactic acid have been shown to inhibit T cell glycolysis
and function (Dröge et al., 1987; Haas et al., 2015). Impaired
glycolysis could relieve GAPDH from its glycolytic function, allow
binding to IFN-g mRNA, and thereby prevent its efficient transla-
tion (Chang et al., 2013). We suggest that intracellular acidifica-
tion also interferes with the regulation of NFAT, an important
transcription factor implicated in the transcriptional control
of IFN-g. Furthermore, acidification may also disturb NFAT
translocation to the nucleus as calcineurin, a phosphatase that
dephosphorylates NFAT and thereby regulates its translocation,
is suppressed by low pH (Hisamitsu et al., 2012).
Moreover, lactic acid concentrations above 20 mM caused T
and NK cell apoptosis, which might explain smaller proportions
of T cells and NK cells in tumors with higher concentrations of
lactate. In contrast, myeloid cells seem to resist the apoptosis-
inducing effect of lactic acid, as high numbers were found in tu-
mors with high lactate levels. Accordingly, we could show that
human monocytes do not undergo apoptosis even in the pres-
ence of high lactic acid concentrations (Dietl et al., 2010). More-
over, lactic acid induces polarization of macrophages to a
M2-like phenotype via induction of hypoxia-inducible factor 1a
(Colegio et al., 2014). In addition, it stimulates expression of
IL-23 (Shime et al., 2008) and increases the number of MDSCs
in the spleens of mice carrying LDHA-expressing tumors (Husain
et al., 2013). It is therefore likely that lactic acid promotes tumor
growth by directly suppressing T cell function and generating tu-
mor-promoting myeloid cells. In support of this theory, we also
detected high numbers of MDSCs, especially in control tumors,
which could promote tumor growth. Production of IFN-g by
T cells and/or NK cells in Ldhalow tumors may counteract
MDSC development, because IFN-g has been shown to support
the reprogramming of MDSCs into antigen-presenting cells (Ker-
kar et al., 2011).
Although lactic acid inhibits T cell and NK cell activation and
leads to MDSC accumulation, we cannot rule out the possibility
that other cells, e.g., B cells, are affected by lactic acid and
contribute to the accelerated tumor growth of B16.SIY control
cells. B lymphocytes have been shown to foster carcinogenesis
in squamous cell carcinoma (Affara et al., 2014), whereas plasma
cell signatures are predictors of favorable survival across solid
tumors in another study (Gentles et al., 2015). Therefore, the
impact of lactic acid on B cells and plasma cells remains to be
elucidated.
 Surprisingly, tumor growth of control and Ldhalow tumors was
similar in Pfp–/– and C57BL/6 mice, suggesting that the granule
exocytosis pathway of T and NK cells is not important for the lim-
itation of tumor growth in Ldhalow tumors. In support of our re-
sults, B16.SIY cells are rarely killed by CD8+ T cells because
PD-L1 limits T cell activity (Blank et al., 2004).
Checkpoint inhibition by PD-1/PD-L1 or CTLA-4 blockade is a
major advance in melanoma therapy; however, only about half
of the patients benefit from this new treatment. Accumulation
of lactic acid in the tumor environment may limit the success of
checkpoint inhibition. Accordingly, blocking acidification prior
to immunotherapy improved the anti-tumor response (Calcinotto
et al., 2012; Pilon-Thomas et al., 2016). In line, two studies
showed that LDH is a selection criterion for ipilimumab and
anti-PD-1 therapy (Diem et al., 2016; Kelderman et al., 2014).
Alternatively, genetic manipulation can rescue T cell function in
a highly glycolytic tumor environment (Ho et al., 2015).
In summary, the (tumor) microenvironment modulates the acti-
vation of infiltrating cells and thereby regulates the balance be-
tween the immune response and tolerance. The finding that
tumor-derived lactic acid can suppress the T and NK cells
adds another piece to the tumor immunosuppression puzzle.
EXPERIMENTAL PROCEDURES
Human Skin Biopsies
Snap-frozen biopsies from tumor and healthy tissue of melanoma patients
were obtained after informed consent. The study was approved by the Institu-
tional Ethics Committee of the University Hospital of Regensburg and de-
signed and conducted in accordance with the Declaration of Helsinki. Clinical
characteristics are summarized in Table S1.
Mice and In Vivo Experiments
Animal experiments were performed according to the regulations of the gov-
ernment of Upper Palatinate, Regensburg, Germany. Unless otherwise indi-
cated, male C57BL/6 mice (Charles River), Rag2–/– and Rag2–/–gc–/– mice
(Taconic), Ifng–/– and Ifngr1–/– mice (The Jackson Laboratory), and Pfp–/–
mice (The Jackson Laboratory) were used. To generate tumors in mice, 1 3
104, 1 3 105, or 1 3 106 tumor cells were injected subcutaneously in the dorsal
region of mice. vav-Bcl-2 mice were used for in vitro analysis of T cells.
Genetic Modulation of Ldha in Tumor Cell Lines
B16.SIY mouse melanoma cells (provided by Christian Blank; Blank et al.,
2004) were transfected with shRNA plasmids (GeneClip U1 Hairpin Cloning
System, Promega) to specifically knockdown expression of Ldha (Ldhalow
clones). Untransfected cells (WT) and cells transfected with a non-specific,
scrambled shRNA (Control) served as controls.
In the mouse pancreatic adenocarcinoma cell line Panc02-H7, Ldha
knockout was performed with CRISPR/Cas9 using a pSpCas9(BB)-2A-GFP
plasmid (Addgene) designed by Jerome Durivault (Centre Scientifique de
Monaco). Untransfected cells (WT) served as controls. Tumor cells were
cultured under standard conditions.
Proliferation
Proliferation of cells was determined by measuring 3H-thymidine incorporation
for 24 hr (Hartmann Analytic).
Western Blotting
Whole cell lysates were analyzed according to standard procedures.
Lactate Measurements
Lactate concentrations in supernatants of cells cultured for 24 hr were deter-
mined enzymatically using an ADVIA 1650 instrument (Bayer) and specific re-
agents (Roche).
Induced Metabolic Bioluminescence Imaging of Intra-tissue Lactate
and Glucose Levels
Metabolites were measured in serial tumor cryosections by the imBI method,
as described earlier (Walenta et al., 2002).
In Vitro Analysis of CD8+ T Cells and NK Cells
CD8+ T cells and NK cells were isolated from spleens by magnetic bead sep-
aration (Miltenyi) and cultured under standard conditions. T cells and NK
cells were incubated in the absence or presence of L-lactic acid (Sigma-Al-
drich) or HCl or sodium L-lactate (Sigma-Aldrich). For cytokine and Ifng
mRNA expression analysis, cells were stimulated with 20 ng/ml PMA (Calbio-
chem) and 1 mM Ionomycin (Enzo Biochem) for 3 hr. For NFAT analysis,
PMA/Ionomycin stimulation was performed for 30 min. For coculture exper-
iments, irradiated B16.SIY cells were cultured with or without 2 ng/ml murine
IFN-g (Pepro Tech). After 24 hr, stimulated 2C CD8+ T cells were added for
another 24 hr.
Quantitative Real-Time PCR
Total RNA of B16.SIY tumor cells, homogenized mouse tissues, and 2C CD8+
T cells was obtained using the RNeasy Mini Kit (QIAGEN). Complementary
DNA was synthesized with a M-MLV Reverse Transcriptase kit (Promega)
and amplified by qPCR with the QuantiFast SYBR Green PCR Kit (QIAGEN)
using the Mastercyler Ep Realplex (Eppendorf).
Quantification of Intracellular pH
Intracellular pH in 2C CD8+ T cells after incubation with different concentra-
tions of L-lactic acid (Sigma-Aldrich) for 30 min was assessed by flow cy-
tometry using the pH-sensitive dye carboxy SNARF-1 AM acetate and
pH-controlled buffers (Life Technologies) for calibration. For analyses, ratios
of emission wavelength l1, transmitted by a 585/42BP filter and wavelength
l2, transmitted by a 670LP filter, were calculated.
ATP Measurement
Cellular ATP levels were determined using the ATP determination kit (Life
Technologies) according to the manufacturer’s protocol.
13
C Tracing Experiments
To determine lactate uptake, 2C CD8+ T cells were incubated for 4 hr with
15 mM 13C1 lactate. Cells were harvested with 80% methanol and analyzed
by GC-MS as described in the Supplemental Experimental Procedures.
Flow Cytometry
Cells were stained with fluorochrome-conjugated antibodies. Intracellular
staining for cytokines and NFATc1 was performed using the BD Cytofix/Cy-
toperm Plus Kit (BD Biosciences). Stained cell populations were acquired
by LSR II (Beckton Dickinson) and data analyzed by FlowJo software
(Tree Star).
Database Analyses
Kaplan Meier estimation curves for overall survival of individual metastatic
melanoma patients were generated with the microarray analysis and visualiza-
tion platform R2 (http://r2.amc.nl) by using the ‘‘R2: Tumor Melanoma
Metastatic – Bhardwaj – 44 – fRMA – u133p2’’ dataset. Correlation of LDHA
expression with GZMK and CD25 was determined with the ‘‘R2: Tumor Skin
Cutaneous Melanoma – TCGA – 470 – rsem – tcgars’’ dataset (http://
cancergenome.nih.gov).
Statistical Analysis
Results represent the mean and SEM. Comparison between groups was
performed using paired or unpaired Student’s t test or one-way ANOVA and
Tukey’s post test (*p < 0.05, **p < 0.01, ***p < 0.001). All calculations were
performed using GraphPad Prism 5 Software.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and one table and can be found with this article online at
http://dx.doi.org/10.1016/j.cmet.2016.08.011.
Cell Metabolism 24, 657–671, November 8, 2016 669
AUTHOR CONTRIBUTIONS
A.B. and K.S. designed experiments, collected data, performed analyses, and
wrote the paper; W.M.-K. and S.W. performed bioluminescence experiments;
G.E.K., M. Kastenberger, G.S., M. Kolitzus, U.S., E.U., R.K., S. Klobuch, S.
Karrer, E.K., S.S., K.P., P.J.O., M.S., K.D., A.T., C.M., C. Bruss, S.H., and
K.R. were involved in experiments and data collection; S.F.-F., C. Blank,
A.M., W.H., J.P., M.B., B.S., E.K.G., and M.M. provided reagents and dis-
cussed data; U.R., C. Bogdan, A.V., and P.H. provided mice and discussed
data; A.S. analyzed data; and M. Kreutz designed the experiments, analyzed
data, and wrote the paper.
ACKNOWLEDGMENTS
We acknowledge the excellent technical assistance of Alice Peuker, Stephanie
Faerber, Monika Wehrstein, and Anna Hoehn. We thank Eva Gottfried for help-
ful discussions, Fabian Schuler for mouse manipulation, and J. Adams for vav-
Bcl-2 mice. This work was supported by DFG KFO 262 and the Mildred Scheel
Foundation (Grants MS 110703 and MS 109247). The results shown here are in
part based upon data generated by The Cancer Genome Atlas Research
Network (http://cancergenome.nih.gov/).
Received: October 16, 2015
Revised: April 20, 2016
Accepted: August 19, 2016
Published: September 15, 2016
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