Expansion and activation of distinct central memory T lymphocyte subsets in complex regional pain syndrome

Abstract

Background: Complex regional pain syndrome (CRPS) is a debilitating condition where trauma to a limb results in devastating persistent pain that is disproportionate to the initial injury. The pathophysiology of CRPS remains unknown; however, accumulating evidence suggests it is an immunoneurological disorder, especially in light of evidence of auto-antibodies in ~ 30% of patients. Despite this, a systematic assessment of all circulating leukocyte populations in CRPS has never been performed.

Methods: We characterised 14 participants as meeting the Budapest clinical criteria for CRPS and assessed their pain ratings and psychological state using a series of questionnaires. Next, we performed immunophenotyping on blood samples from the 14 CRPS participants as well as 14 healthy pain-free controls using mass cytometry. Using a panel of 38 phenotypic and activation markers, we characterised the numbers and intracellular activation status of all major leukocyte populations using manual gating strategies and unsupervised cluster analysis.

Results: We have shown expansion and activation of several distinct populations of central memory T lymphocytes in CRPS. The number of central memory CD8⁺ T cells was increased 2.15-fold; furthermore, this cell group had increased phosphorylation of NFkB and STAT1 compared to controls. Regarding central memory CD4⁺ T lymphocytes, the number of Th1 and Treg cells was increased 4.98-fold and 2.18-fold respectively, with increased phosphorylation of NFkB in both populations. We also found decreased numbers of CD1c⁺ myeloid dendritic cells, although with increased p38 phosphorylation. These changes could indicate dendritic cell tissue trafficking, as well as their involvement in lymphocyte activation.

Conclusions: These findings represent the first mass cytometry immunophenotyping study in any chronic pain state and provide preliminary evidence of an antigen-mediated T lymphocyte response in CRPS. In particular, the presence of increased numbers of long-lived central memory CD4⁺ and CD8⁺ T lymphocytes with increased activation of pro-inflammatory signalling pathways may indicate ongoing inflammation and cellular damage in CRPS.

Keywords: Central memory T lymphocytes; Complex regional pain syndrome; Mass cytometry; Myeloid dendritic cells; NFkB.

Fig. 1

A flow diagram representing the manual gating strategy used to isolate major immune cell populations from mass cytometry output. Firstly, quality control gates were performed to remove normalisation beads, isolate singlets and gate CD45⁺ leukocytes. Next, mononuclear leukocytes (CD66⁻CD61b⁻CD235⁻) were isolated by gating out platelets, granulocytes and erythrocytes. Major lymphocyte and myeloid populations were then isolated using standard phenotypic markers. For example, CD19⁺ for B lymphocytes, CD3⁺ for T lymphocytes, CD56⁺ for NK cells and HLA-DR⁺ for myeloid cells. Finally, major cell populations were gated further into known subsets

Fig. 2

Increased numbers of central memory CD8⁺ T lymphocytes with increased pro-inflammatory activation are found in the blood of CRPS participants compared to healthy controls. a A bar graph showing the number of cells in FlowSoM clusters representing the four major CD8⁺ T lymphocyte populations; naive (Tn, CD3⁺CD8⁺CD45RA⁺CCR7⁺), effector (Teff, CD3⁺CD8⁺CD45RA⁺CCR7⁻), effector memory (Tem, CD3⁺CD8⁺CD45RO⁺CCR7⁻), central memory (Tcm, CD3⁺CD8⁺CD45RO⁺CCR7⁺). b Intracellular signalling and functional marker expression levels in the FlowSoM cluster representing central memory CD8⁺ T lymphocytes. All data are presented as group means (± S.E.M.). * represents P < 0.05 in an unpaired Student’s T Test. c Linear correlation between the number of central memory CD8⁺ T and stress scores on DASS21 in the CRPS group. N = 14 CRPS and N = 14 controls. * represents P < 0.05 in a linear regression analysis

Fig. 3

Increased numbers of central memory CD4⁺ T lymphocytes with increased pro-inflammatory activation are found in the blood of CRPS participants compared to healthy controls. a A bar graph showing the number of cells in FlowSoM clusters representing the four major CD4⁺ T lymphocyte populations; naive (Tn, CD3⁺CD4⁺CD45RA⁺CCR7⁺), effector (Teff, CD3⁺CD4⁺CD45RA⁺CCR7⁻), effector memory (Tem, CD3⁺CD4⁺CD45RO⁺CCR7⁻), central memory (Tcm, CD3⁺CD4⁺CD45RO⁺CCR7⁺). b A bar graph showing the number of cells in FlowSoM clusters representing central memory Th1 lymphocytes (CD3⁺CD4⁺CD45RO⁺CCR7⁺T-bet⁺) and Tregs (CD3⁺CD4⁺CD45RO⁺CCR7⁺CD127^loCD25⁺FoxP3⁺). Intracellular signalling and functional marker expression levels in FlowSoM clusters representing c Th1 lymphocytes and d Tregs. N = 14 CRPS and N = 14 controls. All data are presented as group means (± S.E.M.). * represents P < 0.05 and ** represents P < 0.01 in an unpaired Student’s T Test, and ^# represents P < 0.01 in a Mann-Whitney U Test

Fig. 4

Spanning tree progression of density-normalised events (SPADE) trees showing evidence of pro-inflammatory activation in distinct lymphocyte and myeloid cell populations in the blood of CRPS participants relative to healthy controls. The SPADE algorithm was run on a downsampled population of single leukocytes from all CRPS and control participants. SPADE trees were generated showing the fold-change between CRPS and control groups in the expression of phosphorylated (activated), a p65 NFκB, b AKT, c ERK, d MAPKAPK2, e p38, f PLCγ2, g STAT1, h STAT3 and i STAT5. The major cell populations labelled in (a) are representative of all SPADE trees. Note: colour scales vary for each marker

Fig. 5

Reduced CD1c⁺ myeloid dendritic cell numbers but with increased p38 activation are found in the blood of CRPS participants compared to healthy controls. a A bar graph showing the number of cells in FlowSoM clusters representing the three major dendritic cell populations; CD1c⁺ mDCs (HLA-DR⁺CD14⁻CD11c⁺CD1c⁺CD141⁻), CD141⁺ mDCS (HLA-DR⁺CD14⁻CD11c⁺CD141⁺CD1c⁻) and plasmacytoid DCS (pDCs, HLA-DR⁺CD14⁻CD123⁺) in CRPS and control participants. b Intracellular signalling and functional marker expression levels in the FlowSoM cluster representing CD1c⁺ mDCs. N = 14 CRPS and N = 14 controls. All data are presented as group means (± S.E.M.). ^## represents P < 0.01 in a Mann-Whitney U Test

Fig. 6

Increased pp38 signalling in CD1c⁺ myeloid dendritic cells correlates with increased signalling in T lymphocytes in the blood of CRPS participants. a Linear correlation between pp38 in CD1c + mDCs and pSTAT1 in central memory CD8⁺ T lymphocytes. b Linear correlation between pp38 in CD1c + mDCs and pP65 in central memory CD4⁺ Th1 lymphocytes. * represents P < 0.01 in a linear regression analysis