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Homeostasis and function of T cells in healthy individuals and renal transplant
recipients
Havenith, S.H.C.
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Havenith, S. H. C. (2012). Homeostasis and function of T cells in healthy individuals and renal transplant
recipients
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Download date: 19 Jun 2017
3
CXCR5+CD4+ follicular helper T cells
accumulate in resting human
lymph nodes and have superior
B cell helper activity
Simone H.C. Havenith*,†, Ester B.M. Remmerswaal*,†, Mirza M. Idu‡,
Karlijn A.M.I. van Donselaar - van der Pant †, Nelly van der Bom*,
Fréderike J. Bemelman†, Ester M.M. van Leeuwen*, Ineke J. M. ten Berge†,
and René A. W. van Lier§
Department of Experimental Immunology, Academic Medical Center,
Amsterdam, the Netherlands
†
Renal Transplant Unit, Department of Internal Medicine
and ‡ Department of Surgery, Academic Medical Center, Amsterdam, the Netherlands,
§
Sanquin Research and Landsteiner Laboratory, Academic Medical Centre,
Amsterdam, the Netherlands.
*
45
CD4 + T cells in human lymph nodes
Abstract
Although many relevant immune reactions are initiated in the lymph nodes, this
compartment has not been systematically studied in humans. Analyses have been
performed on immune cells derived from tonsils, but as this tissue is most often
inflamed, generalization of these data is difficult. Here, we analyzed the phenotype
and function of the human CD4+ T cell subsets and lineages in paired resting lymph
node and peripheral blood samples. Naïve, central memory and effector memory cells
as well as Th1, Th2, Th17 and Tregs were equally represented in both compartments.
On the other hand, cytotoxic CD4+ T cells were strikingly absent in the lymph
nodes. CXCR5+CD4+ T cells, representing putative follicular T helper (Tfh) cells were
overrepresented in lymph nodes and expressed higher levels of Tfh markers than their
peripheral blood counterparts. Compared to the circulating pool, lymph node derived
CXCR5+CD4+ T cells were superior in providing help to B cells.
Thus, functionally competent Tfh cells accumulate in resting human lymph nodes
providing a swift induction of naïve and memory antibody responses upon antigenic
challenge.
3
46
CD4 + T cells in human lymph nodes
Introduction
3
CD4+ T cells play a critical role in forming adaptive immune responses against various
pathogens 1-3. Furthermore, they are known to be involved in the pathogenesis of
autoimmune diseases, asthma and allergic responses 4;5. CD4+ T cells consist of
several different T helper (Th) lineages, including Th1, Th2, Th17 and regulatory T
cells (Tregs) 6;7. Additionally, in CMV-infected individuals cytotoxic CD4+ T cells are
found in the circulation 8-10. In recent years, follicular T helper cells (Tfh) have been
identified as the CD4+ T cell subset specialized in supporting activation, expansion
and differentiation of B cells and they are required for the formation of germinal
centers (GCs) 11;12. Expression of CXCR5 and downregulation of CCR7 allows these
cells to migrate from the T cell zone to the primary and secondary follicles, where
they encounter antigen primed B cells 13;14. Human tonsillar Tfh cells express high
initiate levels of ICOS, CD40L, and PD-1 15;16 and the transcription factor BCL-6 17;18
and they produce the B-helper cytokine IL-21 19. In the human peripheral blood
(PB) CXCR5+CD4+ T cells have a CCR7+ central memory phenotype. However, these
circulating CXCR5+CD4+ T cells lack the expression of ICOS, PD-1 and BCL-6 20.
Although adaptive immune responses are initiated in the lymph nodes (LN),
human CD4+ T cells and their different lineages have only been thoroughly studied
in the peripheral blood (PB), also in relation to disease 21-23. The fact that the human
secondary lymphoid compartment is rarely studied can be attributed to the difficulty in
attaining lymphoid tissue. Tonsils, which are surgically removed because of recurrent or
chronic inflammation, have been utilized in the past to study the secondary lymphoid
compartment. However, because of the chronic inflammatory conditions, these studies
likely reflect a skewed image. Data on the different Th lineages and in particular on Tfh
cells in resting human LNs are still lacking.
In the present study, we had the unique opportunity to study CD4+ T cell subsets
in paired human LN and PB samples. We studied the presence of the different
CD4+ T cell subsets and lineages in the LNs and compared this to their presence
in the PB compartment. Furthermore, we analysed phenotypical, but also functional
characteristics of CXCR5+ cells from human LN in comparison to the PB.
Materials and methods
Subjects
We studied paired heparinized peripheral blood mononuclear cells (PBMC) and lymph
node mononuclear cells (LNMC), which were isolated prior to, respectively during
living donor kidney transplantation. EBV- and CMV-serostatus of the patients and their
ages are set out in supplemental table 1. All patients were treated with quadruple
immunosuppression, consisting of CD25mAb induction therapy, prednisolone,
calcineurin-inhibitor and mycophenolic acid. Except for CD25mAb, immunosuppressive
treatment was started immediately after transplantation. At the time the lymph nodes
were gathered, the first dose of CD25mAb was already administered. However, we
47
CD4 + T cells in human lymph nodes
have demonstrated that CD25mAb (basiliximab, Novartis Pharma, Basel, Switzerland)
was not detectable in LNMC and that CD25 expression on LNMC could ex vivo be
blocked with CD25mAb 24. The Medical Ethics Committee of the Academic Medical
Center, Amsterdam, approved the study and all subjects gave written informed consent.
Isolation of mononuclear cells from peripheral blood and lymph nodes
PBMCs were isolated using standard density gradient centrifugation as described
previously 25. Lymph nodes were collected from kidney transplant recipients during
living donor kidney transplantation. Briefly, LNMCs were isolated from surgical residual
material of the recipient, gathered during the implantation of the transplanted kidney.
Before anastomising the arteria and vena renalis, the iliac artery and vein are dissected
free. The residual tissue removed in this procedure to obtain adequate vascular
exposure, often contains lymph nodes. Directly after extraction, the gathered lymph
nodes were chopped in small pieces. A cell suspension was obtained by grinding
the material through a flow-through chamber. PBMCs and LNMCs were subsequently
cryopreserved until the day of analysis.
Virological analysis
To determine CMV serostatus, anti-CMV immunoglobulin G (IgG) was measured in
serum using the AxSYM microparticle enzyme immunoassay (Abbott Laboratories,
Abbott Park, IL, USA) according to the manufacturer’s instructions. Measurements
were calibrated relative to a standard serum. EBV serostatus was determined by
qualitative measurement of specific immunoglobulin G (IgG) against the viral capsid
antigen (VCA) and against nuclear antigen of EBV using respectively the anti-EBV VCA
IgG enzyme-linked immunosorbent assay and the anti-EBV nuclear antigen of EBV
IgG enzyme-linked immunosorbent assay (Biotest, Dreieich, Germany). All tests were
performed following the instructions of the manufacturers.
Immunofluorescence staining; flow cytometry
PBMCs and LNMCs were washed in phosphate-buffered saline (PBS) containing
0.01% NaN3 and 0.5% bovine serum albumin. Half a million PBMCs and LNMCs were
incubated with fluorescence-labeled monoclonal antibodies (mAbs) for 30 minutes
at 4°C, protected from light, at concentrations according to the manufacturer’s
instructions. The following surface antibodies were used: CD8 V450, CD3 V500, CCR7
PE-Cy7, CD25 PE, CXCR5 Alexa Fluor 488, CXCR5 Alexa Fluor 647, ICOS PE (BD
Biosciences, San Jose, CA, USA), CD27 APC-Alexa Fluor 780, CD4 PerCP-eFluor 710,
CD45RA eFluor 605NC, PD1 PE (eBioscience Inc, San Diego, CA, USA), CX3CR1
FITC, CCR6 Alexa Fluor 647, CD69 Alexa Fluor 700 (BioLegend, San Diego, CA,
USA), CXCR3 PE (R&D Systems, Abingdon, UK). For intracellular stainings, cells were
fixed after surface staining with FACS Lysing Solution (BD Biosciences). Following
permeabilization (FACS Permeabilizing Solution 2 (BD Biosciences)) cells were stained
with Ki-67 FITC (BD Biosciences) or granzyme B PE (Sanquin) or granzyme K FITC
(Immunotools, Friesoythe, Germany). For intracellular FOX-P3 stainings, cells were
3
48
CD4 + T cells in human lymph nodes
3
fixed and permeabilized using the Foxp3 Staining Buffer Set and subsequently stained
with Foxp3 APC (both eBioscience). Live/Dead fixable red cell stain kit (Invitrogen,
Paisley, UK) was used in every staining to exclude dead cells from the analysis. Cells
were measured on a LSR-Fortessa flow cytometer (BD Biosciences) and analyzed with
FlowJo software (FlowJo, Ashland, OR, USA)
Detection of cytokines
Cytokine release after peptide or PMA/Ionomycin stimulation was performed as
described by Lamoreaux et al 26, PBMCs and LNMCs were thawed and rested overnight
in suspension flasks (Greiner) in RPMI supplemented with 10% FCS, penicilline and
streptomycine (culture medium). Half a million cells were stimulated for 4 hours at 37ºC
5% CO2 with or without PMA (10ng/ml) and Ionomycin(1μg/ml) in culture medium,
in the presence of αCD28 (15E8) (2μg/ml), αCD29 (TS 2/16) (1 μg/ml) (Sanquin,
Amsterdam, The Netherlands), brefeldin A (Invitrogen) (10μg/ml) and Golgistop (BD
Biosciences) in a final volume of 200μl. Stimulations were performed in non-treated
round bottom 96-well plates (Corning). Subsequently, cells were washed twice and
incubated with CD3 V500, CD8 V450, CD4 PE-Cy5.5 and Live/Dead fixable red cell
stain for 30 min at 4ºC. Then, they were washed twice, fixed and permeabilized
(Cytofix/Cytoperm reagent, BD) and subsequently incubated with a combination of
the following intracellular mAbs: anti-IFNγ APC-Alexa Fluor 750 (Invitrogen), -IL-4
PE-Cy7, -IL-17A APC-Alexa Fluor 780, -IL-21 Alexa Fluor 647 (eBioscience), -IL-2 PE,
-IL-5 PE (BioLegend) for 30 min at 4ºC. Cells were washed twice and measured on a LSR
Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (FlowJo).
Cell sorting
For isolation of CXCR5+ memory T cells, cells were labeled with CD3 PE (eBioscience),
CD4 APC, CD45RA PE Cy7, CXCR5 AF488 (BD Biosciences) and CCR7 PerCPCy5.5
(BioLegend). For isolation of B cells, cells were labeled with CD19 PE and CD20 APC
(BD Biosciences). Cells were sorted on a FACSARIA (BD Biosciences). Purity of the
obtained sorted cells was verified by flowcytometry and was at least 95%.
B cell co-culture and stimulation
Sorted LN and PB derived CXCR5+CD4+ T cells were co-cultured with LN and PB
derived B cells for 7 days. Cells were cultured in RPMI (Invitrogen) containing 10%
fetal calf serum, 100U/ml sodium penicillin G (Brocades Pharma BV, Leiderdorp, The
Netherlands) and 100ug/ml streptomycin sulphate (Invitrogen). For stimulation we
added soluble 1ug/ml anti-CD3 (clone 1XE) and soluble 5ug/ml anti-CD28 (clone
15E8) to the cell culture.
ELISA IgG IgM
Supernatants were tested for secreted IgM and IgG with an in-house ELISA using
polyclonal rabbit anti-human IgG and IgM reagents and a serum protein calibrator all
from Dako (Heverlee, Belgium), as described previously in more detail 27.
49
CD4 + T cells in human lymph nodes
Statistical analysis
Statistical analysis of paired samples was done by two tailed Wilcoxon signed rank test
with a 95% confidence interval.
Results
LN and PB contain equal percentages of naïve, central memory and effector
memory CD4+ T cells
We collected paired LN and PB samples in which we compared the presence of total
CD4+ T cells and of naïve, central-memory and effector-memory CD4+ T cells. The
percentage of CD4+ T cells within the CD3+ subset was similar in LNs and PB (figure 1A).
We phenotypically distinguished naïve, central memory and effector memory CD4+
T cells based upon their expression of CCR7 and CD45RA 28. We demonstrated that
CCR7+CD45RA+ naïve (Tn), CCR7+CD45RA- central memory (Tcm) and CCR7- effector
memory (Tem) CD4+ T cells were equally represented in LN and PB (figure 1B).
Th1, Th2, Th17 and Tregs are evenly divided over the LN and PB compartment
To learn more about the distribution of the different T helper cell lineages over LN and
PB, we analysed the chemokine-receptor expression pattern and cytokine producing
capacity of the CD4+ T cells in both compartments. The chemokine-receptors CXCR3
and CCR6 can help distinguish between Th1, Th2 and Th17 cells 29-33. We found
that the percentages of CXCR3+CCR6- Th1, CXCR3-CCR6- Th2 and CXCR3-CCR6+
Th17 memory CD4+ T cells were equal in PB and LN (figure 2A and B). Markedly, the
Figure 1) CD4+ T cells are equally represented in LN and PB. (A) CD4+ T cells as a
percentage of total T cells in LN compared to PB. (B) Percentage of CCR7+CD45RA+ naïve
(Tn), CCR7+CD45RA- central memory (Tcm) and CCR7-CD45RA- effector memory (Tem)
CD4+ T cells within total CD4+ T cells in LN compared to PB. PB and LN are paired samples,
derived from the same patient. Statistical analysis was done using Wilcoxon signed rank
test. Data are representative for 17 individuals.
3
50
CD4 + T cells in human lymph nodes
3
percentage of CXCR3-CCR6- Th2 cells in the PB showed a strong correlation with the
percentage of Th2 cells present in the LN (figure 2C), whereas the percentage of Th1
and Th17 cells in PB and LN did not correlate significantly (figure 2D and E). Next,
we functionally discriminated Th cells according to their cytokine producing capacity.
PB CD4+ T cells produced significantly more IFNγ (p=0.003), while IL-2 was equally
expressed by both LN and PB CD4+ T cells (figure 2F and G). The percentage of CD4+
T cells producing both IL-4 and IL-5, although very low, was significantly higher in
PB (p=0.008; figure 2H and I). The percentage of IL-5 single positive cells was also
significantly higher in the PB (p=0.006), while IL-4 single positive cells were equally
present in LN and PB (data not shown). IL-17 was produced in similar percentages by
PB and LN CD4+ T cells (figure 2J and K).
Finally, we questioned how the percentage of LN regulatory CD4+ T cells relates to their
percentage in the PB. Therefore, we analysed FoxP3, CD25 and IL-7Rα (CD127) expression
on both LN and PB derived CD4+ T cells. We found that FoxP3+CD25brightCD127low Tregs
were equally distributed between LN and PB (figure 3A and B). The amount of Tregs in the
PB did not correlate with the amount in the LNs (figure 3C).
Overall, these data imply that the different Th lineages, consisting of Th1, Th2, Th17
and Tregs are equally distributed over PB and LN. However, IFNγ and IL-4/IL-5 double
producing CD4+ T cells were somewhat over-represented in the PB compartment.
Cytotoxic, granzyme B+ CD4+ T cells are not detectable in the LN
Latent infection with cytomegalovirus (CMV) has a great impact on the composition
of the PB CD8+ T cell compartment but not on the LN CD8+ T cell compartment 24.
We studied cytotoxic, granzyme B+, CD4+ T cells which have been shown to appear
as a consequence of CMV infection 9;10;34. In the PB, cytotoxic CD4+ T cells are
characterized by the lack of expression of the costimulatory molecules CD27 and
CD28 9. Furthermore, they express CX3CR1 35, which provides them with the capability
to migrate to inflamed vascular endothelium. We found that CD27-CD28- (figure 4A
and B), CX3CR1+CCR7- (figure 4C and D) and granzyme B+ (figure 4E and F) CD4+ T
cells were not at all detectable in LNs.
CXCR5+CD4+ T cells accumulate in LNs and express higher levels of Tfh-markers as
compared to their PB counterparts
Tfh cells express CXCR5 and are specialized in providing help to B cells in the LN. In
humans, but not in mice, CXCR5 expressing CD4+ T cells are also found in the PB. The
percentage of CXCR5 expressing CD4+ T cells in resting LN (mean 27%, SD ±13%)
was significantly higher than in the circulation (mean 12%, SD ±5%) (figure 5A).
Interestingly, similar to Th2 cells, the percentage of CXCR5+CD4+ T cells showed a
strong correlation between PB and LN (figure 5B). LN CXCR5+CD4+ T cells expressed
significantly more ICOS (figure 5C and H) and PD-1 (figure 5D and I) than their PB
counterparts. Furthermore, LN CXCR5+CD4+ T cells expressed significantly less CCR7
(figure 5E and J) and more CD69 as compared to PB derived CXCR5+CD4+ T cells
(figure 5F and K). IL-21 is a cytokine known to play an important role in class switch
51
CD4 + T cells in human lymph nodes
3
Figure 2) LN and PB contain equal percentages of CD4+ T cell lineages Th1, Th2, Th17. (A)
Comparison of CXCR3 (x-axis) and CCR6 (y-axis) expression on LN and PB memory CD4+
T cells. Dotplots are gated on CD3+CD4+CD45RA- lymphocytes. The left panel represents
the LN and the right panel the PB derived memory CD4+ T cells. (B) Paired analysis of
the percentage of CXCR3+CCR6- (Th1), CXCR3-CCR6- (Th2) and CXCR3-CCR6+ (Th17)
memory CD4+ T cells in LN as compared to PB. Correlation between the percentage of
(C) Th1 CXCR3+CCR6- (R2 = 0.3), (D) Th2 CXCR3-CCR6- (R2=0.56, p= 0.02) and (E) Th17
CXCR3-CCR6+ (R2=0.006) memory CD4+ T cells in LN on the x-axis and PB on the y-axis.
(F) Comparison of IFN-γ (x-axis) and IL-2 (y-axis) expression by LN (left panel) and PB (right
panel) CD4+ T cells. (G) Paired analysis of the percentage of IFNγ (left panel) and IL-2 (right
panel) producing CD4+ T cells in LN compared to PB. (H) Comparison of IL-5 (x-axis) and
IL-4 (y-axis) producing CD4+ T cells in LN (left panel) and PB (right panel). (I) Paired analysis
of the percentage of IL-4/IL-5 double producing CD4+ T cells in LN and PB. (J) IL-17 (x-axis)
producing CD4+ T cells in LN (left panel) as compared to PB (right panel). (K) Paired analysis
of the percentage of IL-17 producing CD4+ T cells. In figures D, F, H and J the dotplots were
gated on CD3+CD4+ lymphocytes. Statistical analysis was done using Wilcoxon signed rank
test. The data are representative for 9 individuals. * = p<0.05, ** = p<0.01
52
CD4 + T cells in human lymph nodes
3
Figure 3) Tregs are equally distributed over LN and PB. (A) Comparison of FoxP3 (x-axis)
and CD25 (y-axis) expression on LN (left panel) and PB (right panel) CD4+ T cells. (B) Paired
analysis of FoxP3+CD25brightCD127dim CD4+ T cells in LN compared to PB. Dotplots were
gated on CD3+CD4+ lymphocytes. Statistical analysis was done using Wilcoxon signed rank
test. The data are representative for 9 individuals. * = p<0.05, ** = p<0.01
recombination, plasma cell differentiation and proliferation of B cells. Indeed, also
IL-21 secreting CD4+ T cells reside more in the LNs (figure 5G and L).
In conclusion, we demonstrated that the percentage of CXCR5+CD4+ T cells was
higher in LNs than in the circulating pool and possessed more Tfh characteristics.
Compared to PB, LN CXCR5+CD4+ T cells provide superior B cell help for Ig production
Previous data have demonstrated that tonsil derived CXCR5+CD4+ T cells exhibit higher
B helper cell activity in vitro as compared to CXCR5-CD4+ T cells 36. Furthermore, PB
CXCR5+ cells have also been shown to induce more Ig production by B-cells as compared
to PB CXCR5-CD4+ T cells 37. However, a paired functional comparison, of PB and LN
derived CXCR5+CD4+ T cells, has not been performed previously. To determine B cell
helper capacity, we sorted CXCR5+CD4+ T cells from both LN and PB and added each of
them in a 1:1 concentration to either sorted LN or PB derived B cells. Subsequently, the
cells were stimulated for 7 days with a TCR stimulus and costimulation. LN CXCR5+CD4+
T cells induced more IgG and IgM production as compared to PB CXCR5+CD4+ T cells
when co-cultured with either LN or PB derived B cells (Figure 6A and B). Furthermore,
LN derived B cells were more potent producers of IgG as compared to PB derived B
cells (Figure 6A and B). With regard to the production of IgM, the difference between
LN and PB B cells was less obvious (Figure 6A and B). As shown in figure 6C and D, LNs
contained more switched memory phenotype CD27+IgD- B cells, which may explain the
difference found in IgG production by LN and PB B cells.
Together, these data show that LN derived CXCR5+CD4+ T cells have superior
capacity to provide help to B cells required for IgG and IgM production.
53
CD4 + T cells in human lymph nodes
3
Figure 4) Cytotoxic CD4+ T cells are excluded from the LNs. Comparison of (A) CD27
(x-axis) and CD28 (y-axis), (C) CX3CR1 (x-axis) and CCR7 (y-axis) and (E) Granzyme K (x-axis)
and Granzyme B (y-axis) expression on LN (left panels) and PB (right panels) CD4+ T cells.
Dotplots are gated on CD3+CD4+ lymphocytes. Paired analysis of the percentage of (B)
CD27-CD28- CD4+ T cells, (D) CCR7-CX3CR1+ CD4+ T cells and (F) Granzyme B+ CD4+ T
cells in LN as compared to PB. Statistical analysis was done using Wilcoxon signed rank
test. Data are representative for 11 individuals. * = p<0.05, ** = p<0.01
54
CD4 + T cells in human lymph nodes
3
Figure 5) CXCR5+CD4+ T cells expressing ICOS, PD-1 and CD69 are over-represented in LN
as compared to the PB compartment. (A) Paired comparative analysis of the percentage of
CXCR5+ cells within LN and PB CD4+ T cells. (B) Correlation of the percentage CXCR5+ cells
within LN (x-axis) and PB (y-axis) CD4+ T cells. Comparison of (C) CXCR5 (x-axis) and ICOS
(y-axis), (D) CXCR5 (x-axis) and PD1 (y-axis), (E) CXCR5 (x-axis) and CCR7 (y-axis), (F) CXCR5
(x-axis) and CD69 (y-axis) expression and (G) IL-21 (x-axis) production in LN (left) and PB
(right). Dotplots are gated on CD3+CD4+ lymphocytes. Paired comparative analysis of the
percentage of (H) ICOS, (I) PD1, (J) CCR7 and(K) CD69 expression on LN and PB CXCR5+
CD4+ T cells. (L) Paired analysis of IL-21 production by LN and PB derived CD4+ T cells.
Statistical analysis was done using Wilcoxon signed rank test. Data are representative for 9
individuals. * = p<0.05, ** = p<0.01, *** = p< 0.001, **** = p<0.0001
55
CD4 + T cells in human lymph nodes
3
Figure 6) LN CXCR5+CD4+ T cells provide superior help to B cells. LN CXCR5+CD4+ T cells
(black bars) and PB CXCR5+CD4+ T cells (gray bars) were co-cultured with LN B cells (A) or
PB B cells (B) and subsequently stimulated with αCD3αCD28. We measured IgG and IgM
in the culture supernatants by means of ELISA. IgG and IgM are expressed in percentages,
where 100% is the highest amount of IgG or IgM produced within each separate individual.
Figure 1A and 1B represent 4 separate experiments. Absolute values ranged from 65 to
3172 ng/ml of IgG and from 82 to 21622 ng/ml of IgM. (C) IgD and CD27 expression on LN
(right panel) and PB (left panel) B cells. Dotplots are gated on CD19+CD20+ lymphocytes
(D) Paired analysis of CD27+IgD- memory B cells in LN compared to PB (n=6). Statistical
paired analysis was done using Wilcoxon signed rank test. Data are representative for 9
individuals. ** = p<0.01
Discussion
Although most adaptive immune responses are initiated in the LNs, data on the distribution
of differentiated Th cells within resting LNs and PB were still lacking. We are the first to
compare the circulating CD4+ T-cell compartment to CD4+ T cells residing in resting LNs.
Overall Th1, Th2, Th17 cells and Tregs were found to be equally spread over the two
compartments, whereas cytotoxic CD4+ T cells were absent from the LNs. Furthermore, the
resting human LN compartment comprised significantly more CXCR5+CD4+ T cells than
the PB compartment. Apart from significantly higher expression levels of ICOS, PD-1 and
CD69 and lower expression of CCR7, these cells also provide stronger helper activity for
Immunoglobulin (Ig) production in vitro than their PB counterparts.
The chemokine-receptor CCR7 and its ligands play an important role in directing
the migration of T cells into the LNs 28. Surprisingly, we found that Tn and Tcm cells,
which both express CCR7, were not enriched in the LNs and that Tem, which lack
CCR7 expression, were also equally present in PB and LN. This might be explained
56
CD4 + T cells in human lymph nodes
3
by the fact that the Tcm cells, once they have reached the LN, down-regulate CCR7
upon contact with its ligand CCL19 (25,26). Moreover, Tem cells might reach the LN
by means of inflammatory chemokines and their receptors, such as CXCL10 and its
receptor CXCR3 38. Based on the low expression of cell activation markers such as
Ki67 and HLA-DR (data not shown), the LNs analyzed in our study are presumably in
a resting state. Still, they originate from the para-iliac region draining the pelvic area
and parts of the intestine, areas which are constantly exposed to pathogens. Thus,
low levels of CXCL10 might continuously be produced in these LNs, attracting CXCR3
expressing T cells from the peripheral blood compartment.
The percentages of both CXCR5+CD4+ T cells and CXCR3-CCR6- Th2 cells show
a strong correlation between PB and LN. Apparently, the frequencies of circulating
CXCR5+CD4+ T cells and Th2 cells are a reflection of the frequencies present in the
LNs. This is in agreement with the fact that LNs are the most important effector site
for both Tfh and Th2 cells. On the other hand the percentages of Th1, Th17 cells and
Tregs do not correlate between LN and PB. The fact that we could not find a correlation
here can perhaps be explained by the fact that Th1, Th17 cells and Tregs are also
distributed over other compartments, such as the spleen, peripheral tissues and/or
sites of infection 39;40. Moreover, they do not have LNs as their primary effector site.
LN and PB samples analysed in this study are derived from patients undergoing
kidney transplantation. We can’t exclude that end stage renal disease (ESRD) might have
influenced our results. Loss of renal function has been associated with decreased responses
to vaccination such as HBV 41. Indeed, patients with ESRD have an increased susceptibility
to infectious diseases and increased mortality caused by sepsis 42. Furthermore, ESRD and
uraemia have been associated with a depletion of naïve and central memory T cells and
B cells 43. Although these findings may point to a defect in T helper function, our in vitro
studies demonstrated proper helper activity of the isolated CD4 populations.
Judging from their chemokine-receptor expression pattern, Th1 and Th2 cells are
equally distributed over the two compartments. However, the Th1-cytokine IFN-γ and the
Th2-cytokines IL-4/IL-5 are produced slightly more by the PB CD4+ T cells. The higher
percentage of IFN-γ producing CD4+ T cells in the PB is possibly due to the fact that the PB
contains more cytotoxic CD28-CD4+ T cells, whereas these cells are absent from the LNs.
We have previously shown that a larger proportion of CD28-CD4+ T cells produce IFN-γ
as compared to CD28+CD4+ T cells 10. The discrepancy between Th1/Th2 chemokinereceptor expression and cytokine production can also be caused by a disproportionate
up- or down-regulation of chemokine-receptors in the different compartments.
Remarkably, we did not detect any cytotoxic GranzymeB+ CD4+ T cells in LNs,
whereas they were present in the PB compartment of CMV seropositive individuals.
Previously, we have established that circulating cytotoxic CD4+ T cells appear as a
consequence of CMV infection 9;10. Like CMV-specific CD8+ T cells, which were also
shown to be mainly present in the PB compartment 24, the cytotoxic CD4+ T cells
express CX3CR1 and lack the expression of CCR7, favoring their migration to CMVinfected vascular endothelium 44-47.
57
CD4 + T cells in human lymph nodes
In humans, several studies have been performed on the presence and function
of CXCR5+CD4+ so-called follicular T helper (Tfh) cells in tonsillar lymphoid tissue.
Functional studies demonstrate that tonsil CXCR5+CD4+ T cells are superior in
supporting Ig production as compared to tonsil CXCR5-CD4+ T cells 36. The same holds
true for PB CXCR5+ as compared to CXCR5-CD4+ T cells 37;48. Because of the scarce
accessibility of LNs for human research, until now, no studies on CXCR5+CD4+ T cells
in resting LN have been performed. We are the first to demonstrate that CXCR5+CD4+
T cells expressing Tfh cell markers are present in significantly higher numbers in the
LNs than in the PB. Subsequently, we show that LN derived CXCR5+CD4+ T cells are
better at providing B-cell help required for Ig production than their PB counterparts.
When comparing our data to previous studies on tonsil derived CXCR5+CD4+ T cells,
we noticed that the percentage of CXCR5+CD4+ T cells we found in LNs is much lower
than the percentage previously found in human tonsils 15;36;49. This difference might be
explained by the fact that most tonsillectomies are performed because of recurrent or
chronic inflammation, whereas the LNs we studied, are not inflamed. Furthermore, in
human tonsillar tissue, different subsets of Tfh cells have been described: CXCR5hiICOShi
and CXCR5loICOSloCD4+ T cells 49;50. The CXCR5hiICOShiCD4+ T cells express high
amounts of PD-1 and are found in the germinal centers (GCs), where they provide
help to GC-B cells. In contrast, CXCR5loICOSloCD4+ T cells express lower amounts of
PD-1 and have been shown to be Tfh-committed and to help B-cell responses outside
the GCs 49. Likely, CXCR5loICOSloCD4+ T cells function at the T cell – B cell border
during the initial phase of B cell activation and they contain precursors from which
CXCR5hiICOShiCD4+ T cells, GC Tfh cells, emerge in a later stage 12. We did not detect
CXCR5hiICOShiCD4+ T cells in the LNs. Again this might be explained by the fact
that in tonsils an immune response is ongoing, while the LNs studied here represent
lymphoid tissue during homeostatic condition. In a recent study, that primarily focused
on HIV infected individuals, a high percentage of CXCR5+PD-1hi cells in peripheral LNs
is described 51, which may be explained by an ongoing immune response because of
viral persistence in these HIV patients. Increased differentiation toward Tfh cells has
also been observed in LCMV mouse model for persisting viral infection 52. We did find
CXCR5+CD4+ T cells expressing low amounts of ICOS and PD-1 in the LNs while PB
CXCR5+CD4+ T cells do not express ICOS or PD-1. As mentioned earlier, the resting
LNs analyzed in the present study are most likely continuously exposed to a low burden
of pathogens, which may explain the presence of the CXCR5loICOSloCD4+ T cells.
Apparently a limited exposure to pathogens does not induce CXCR5hiICOShiCD4+ T
cells which are necessary for GC reactions.
In conclusion, we show that Th1, Th2, Th17 and Tregs are equally distributed over
LN and PB, while cytotoxic granzymeB+ CD4+ effector T cells are only detectable at
their site of action, namely in the PB compartment. Furthermore, as could be expected
from their capacity to provide help in antibody production, indeed CXCR5+CD4+ T
cells are dominantly present in resting LNs. Apart from that, these cells possess both
functional and phenotypic characteristics that resemble the Tfh cells as characterized
3
58
CD4 + T cells in human lymph nodes
3
in previous studies. Circulating CXCR5+CD4+ T cells are often studied and have been
shown to be increased in various autoimmune diseases 22;37 However, because we
demonstrated that functionally and phenotypically CXCR5+CD4+ T cells derived from
resting LN and PB differ substantially, one should be cautious with the interpretation
of studies analysing only circulating CXCR5+CD4+ T cells.
Reference List
1.
Swain SL, McKinstry KK, Strutt TM.
Expanding roles for CD4(+) T cells in
immunity to viruses. Nat.Rev.Immunol.
2012;12:136-148.
2.
O’Shea JJ, Paul WE. Mechanisms underlying
lineage commitment and plasticity of helper
CD4+ T cells. Science 2010;327:1098-1102.
3.
Gamadia LE, Rentenaar RJ, van Lier RA,
ten Berge IJ. Properties of CD4(+) T cells
in human cytomegalovirus infection. Hum.
Immunol. 2004;65:486-492.
4.
Lloyd CM, Hessel EM. Functions of T cells
in asthma: more than just T(H)2 cells. Nat.
Rev.Immunol. 2010;10:838-848.
5.
Goverman J. Autoimmune T cell responses
in the central nervous system. Nat.Rev.
Immunol. 2009;9:393-407.
6.
Zhu J, Yamane H, Paul WE. Differentiation
of effector CD4 T cell populations (*).
Annu.Rev.Immunol. 2010;28:445-489.
7.
Zygmunt B, Veldhoen M. T helper cell
differentiation more than just cytokines.
Adv.Immunol. 2011;109:159-196.
8.
Appay V, Zaunders JJ, Papagno L et al.
Characterization of CD4(+) CTLs ex vivo.
J.Immunol. 2002;168:5954-5958.
9.
van de Berg PJ, van Leeuwen EM, ten Berge
IJ, van LR. Cytotoxic human CD4(+) T cells.
Curr.Opin.Immunol. 2008;20:339-343.
10. van Leeuwen EM, Remmerswaal EB,
Vossen MT et al. Emergence of a CD4+.
J.Immunol. 2004;173:1834-1841.
11. Deenick EK, Ma CS. The regulation and
role of T follicular helper cells in immunity.
Immunology 2011;134:361-367.
12. Ma CS, Deenick EK, Batten M, Tangye
SG. The origins, function, and regulation
of T follicular helper cells. J.Exp.Med.
2012;209:1241-1253.
13. Ansel KM, McHeyzer-Williams LJ, Ngo
VN, McHeyzer-Williams MG, Cyster JG.
In vivo-activated CD4 T cells upregulate
CXC chemokine receptor 5 and reprogram
their response to lymphoid chemokines.
J.Exp.Med. 1999;190:1123-1134.
14. Hardtke S, Ohl L, Forster R. Balanced
expression of CXCR5 and CCR7 on
follicular T helper cells determines their
transient positioning to lymph node
follicles and is essential for efficient B-cell
help. Blood 2005;106:1924-1931.
15. Schaerli P, Willimann K, Lang AB et al. CXC
chemokine receptor 5 expression defines
follicular homing T cells with B cell helper
function. J.Exp.Med. 2000;192:1553-1562.
16. Haynes NM, Allen CD, Lesley R
et al. Role of CXCR5 and CCR7 in
follicular Th cell positioning and
appearance of a programmed cell
death gene-1high germinal centerassociated subpopulation. J.Immunol.
2007;179:5099-5108.
17. Johnston RJ, Poholek AC, DiToro D et
al. Bcl6 and Blimp-1 are reciprocal and
antagonistic regulators of T follicular
helper cell differentiation. Science
2009;325:1006-1010.
18. Kroenke MA, Eto D, Locci M et al. Bcl6
and Maf cooperate to instruct human
follicular helper CD4 T cell differentiation.
J.Immunol. 2012;188:3734-3744.
19. Chtanova T, Tangye SG, Newton R et al. T
follicular helper cells express a distinctive
transcriptional profile, reflecting their role as
non-Th1/Th2 effector cells that provide help
for B cells. J.Immunol. 2004;173:68-78.
20. Forster R, Emrich T, Kremmer E, Lipp M.
Expression of the G-protein--coupled
receptor BLR1 defines mature, recirculating
B cells and a subset of T-helper memory
cells. Blood 1994;84:830-840.
21. Beyer M, Schultze JL. Regulatory T cells in
cancer. Blood 2006;108:804-811.
22. Saito R, Onodera H, Tago H et al. Altered
expression of chemokine receptor CXCR5
59
CD4 + T cells in human lymph nodes
on T cells of myasthenia gravis patients.
J.Neuroimmunol. 2005;170:172-178.
23. Tesmer LA, Lundy SK, Sarkar S, Fox DA.
Th17 cells in human disease. Immunol.
Rev. 2008;223:87-113.
24. Remmerswaal EB, Havenith SH, Idu MM
et al. Human virus-specific effector-type T
cells accumulate in blood but not in lymph
nodes. Blood 2012;119:1702-1712.
25. van de Berg PJ, Griffiths SJ, Yong SL et
al. Cytomegalovirus infection reduces
telomere length of the circulating T cell
pool. J.Immunol. 2010;184:3417-3423.
26. Lamoreaux L, Roederer M, Koup R.
Intracellular cytokine optimization and
standard operating procedure. Nat.
Protoc. 2006;1:1507-1516.
27. Kuijpers TW, Bende RJ, Baars PA et al. CD20
deficiency in humans results in impaired
T cell-independent antibody responses.
J.Clin.Invest 2010;120:214-222.
28. Sallusto F, Lenig D, Forster R, Lipp M,
Lanzavecchia A. Two subsets of memory
T lymphocytes with distinct homing
potentials and effector functions. Nature
1999;401:708-712.
29. Maecker HT, McCoy JP, Nussenblatt R.
Standardizing immunophenotyping for
the Human Immunology Project. Nat.Rev.
Immunol. 2012;12:191-200.
30. Annunziato F, Cosmi L, Santarlasci V et
al. Phenotypic and functional features
of human Th17 cells. J.Exp.Med.
2007;204:1849-1861.
31. Bonecchi R, Bianchi G, Bordignon PP et
al. Differential expression of chemokine
receptors and chemotactic responsiveness
of type 1 T helper cells (Th1s) and Th2s.
J.Exp.Med. 1998;187:129-134.
32. Sallusto F, Lenig D, Mackay CR,
Lanzavecchia A. Flexible programs of
chemokine receptor expression on human
polarized T helper 1 and 2 lymphocytes.
J.Exp.Med. 1998;187:875-883.
33. Singh SP, Zhang HH, Foley JF, Hedrick
MN, Farber JM. Human T cells that
are able to produce IL-17 express the
chemokine receptor CCR6. J.Immunol.
2008;180:214-221.
34. van de Berg PJ, van SA, ten Berge IJ, van Lier
RA. A fingerprint left by cytomegalovirus
infection in the human T cell compartment.
J.Clin.Virol. 2008;41:213-217.
35. van de Berg PJ, Yong SL, Remmerswaal EB,
van Lier RA, ten Berge IJ. Cytomegalovirusinduced effector T cells cause endothelial
cell damage. Clin.Vaccine Immunol.
2012;19:772-779.
36. Breitfeld D, Ohl L, Kremmer E et al.
Follicular B helper T cells express CXC
chemokine receptor 5, localize to B cell
follicles, and support immunoglobulin
production. J.Exp.Med. 2000;192:15451552.
37. Morita R, Schmitt N, Bentebibel SE et al.
Human blood CXCR5(+)CD4(+) T cells
are counterparts of T follicular cells and
contain specific subsets that differentially
support antibody secretion. Immunity.
2011;34:108-121.
38. Guarda G, Hons M, Soriano SF et al.
L-selectin-negative.
Nat.Immunol.
2007;8:743-752.
39. Mosser DM. The many faces of macrophage
activation. J.Leukoc.Biol. 2003;73:209-212.
40. Ouyang W, Kolls JK, Zheng Y. The
biological functions of T helper 17 cell
effector cytokines in inflammation.
Immunity. 2008;28:454-467.
41. DaRoza G, Loewen A, Djurdjev O et
al. Stage of chronic kidney disease
predicts seroconversion after hepatitis
B immunization: earlier is better.
Am.J.Kidney Dis. 2003;42:1184-1192.
42. Sarnak MJ, Jaber BL. Mortality caused
by sepsis in patients with end-stage
renal disease compared with the general
population. Kidney Int. 2000;58:1758-1764.
43. Vaziri ND, Pahl MV, Crum A, Norris K. Effect of
uremia on structure and function of immune
system. J.Ren Nutr. 2012;22:149-156.
44. Bolovan-Fritts CA, Spector SA. Endothelial
damage from cytomegalovirus-specific
host immune response can be prevented by
targeted disruption of fractalkine-CX3CR1
interaction. Blood 2008;111:175-182.
45. Umehara H, Bloom ET, Okazaki T et al.
Fractalkine in vascular biology: from basic
research to clinical disease. Arterioscler.
Thromb.Vasc.Biol. 2004;24:34-40.
46. Grefte JM, van der Giessen M, Blom
N, The TH, van Son WJ. Circulating
cytomegalovirus-infected
endothelial
cells after renal transplantation: possible
clue to pathophysiology? Transplant.Proc.
1995;27:939-942.
3
60
CD4 + T cells in human lymph nodes
3
47. Sinzger C, Grefte A, Plachter B et al.
Fibroblasts, epithelial cells, endothelial
cells and smooth muscle cells are major
targets of human cytomegalovirus infection
in lung and gastrointestinal tissues. J.Gen.
Virol. 1995;76 ( Pt 4):741-750.
48. Chevalier N, Jarrossay D, Ho E et al.
CXCR5 expressing human central memory
CD4 T cells and their relevance for
humoral immune responses. J.Immunol.
2011;186:5556-5568.
49. Bentebibel SE, Schmitt N, Banchereau J,
Ueno H. Human tonsil B-cell lymphoma
6 (BCL6)-expressing CD4+ T-cell subset
specialized for B-cell help outside
germinal
centers.
Proc.Natl.Acad.
Sci.U.S.A 2011;108:E488-E497.
50. Rasheed AU, Rahn HP, Sallusto F, Lipp M,
Muller G. Follicular B helper T cell activity is
confined to CXCR5(hi)ICOS(hi) CD4 T cells
and is independent of CD57 expression.
Eur.J.Immunol. 2006;36:1892-1903.
51. Lindqvist M, van LJ, Soghoian DZ et al.
Expansion of HIV-specific T follicular
helper cells in chronic HIV infection. J.Clin.
Invest 2012;122:3271-3280.
52. Fahey LM, Wilson EB, Elsaesser H et al.
Viral persistence redirects CD4 T cell
differentiation toward T follicular helper
cells. J.Exp.Med. 2011;208:987-999.
61
CD4 + T cells in human lymph nodes
supplementary material
Supplemental Table 1
patient
Immunosuppressive regimen
CMV
EBV
age
figure
Pt682
P/MMF/FK/CD25mAb
-
+
62
1
Pt701
P/MMF/FK/CD25mAb
-
+
68
1
Pt466
P/MMF/FK/CD25mAb
+
+
47
1,3
Pt498
P/MMF/FK/CD25mAb
+
+
52
1,3
Pt516
P/MMF/FK/CD25mAb
+
+
58
1,3
Pt532
P/MMF/FK/CD25mAb
+
+
41
1,3
Pt560
P/MMF/FK/CD25mAb
+
+
65
1,3
Pt567
P/MMF/FK/CD25mAb
+
+
63
1,3
Pt647
P/MMF/FK/CD25mAb
+
+
26
1,3
Pt651
P/MMF/FK/CD25mAb
+
+
55
1,3
Pt670
P/MMF/FK/CD25mAb
+
+
49
1,3
Pt692
P/MMF/FK/CD25mAb
+
+
65
1,3
Pt524
P/MMF/FK/CD25mAb
-
+
53
1,2,4
Pt719
P/MMF/FK/CD25mAb
+
+
26
1,2,4
Pt753
P/MMF/FK/CD25mAb
-
+
73
1,2,4
Pt797
P/MMF/FK/CD25mAb
+
+
25
1,2,4
Pt813
P/MMF/FK/CD25mAb
-
-
33
1,2,4
Pt827
P/MMF/FK/CD25mAb
-
+
22
1,2,4,5
Pt868
P/MMF/FK/CD25mAb
-
+
58
1,2,4,5
Pt778
P/MMF/FK/CD25mAb
+
-
57
1,2,4,5
PtSi
P/MMF/FK/CD25mAb
+
+
41
1,2,4,5
3