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, 126 (3), 560-9

Characterization of Thoracic Duct Cells That Transfer Polyarthritis

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Characterization of Thoracic Duct Cells That Transfer Polyarthritis

L D Spargo et al. Clin Exp Immunol.

Abstract

Polyarthritis may result from the haematogenous distribution of arthritogenic effector lymphocytes that emerge in the efferent lymph and pass through the thoracic duct (TD) to the circulation. We therefore examined whether TD cells collected from rats in the late prodrome of adjuvant-induced arthritis (AA) could transfer polyarthritis adoptively and whether these cells included a subpopulation of arthritogenic cells that could be identified phenotypically. Unfractionated TD cells collected from donor rats 9 days after adjuvant inoculation were injected intravenously into normal syngeneic recipients in numbers equivalent to the overnight harvest from a single donor. TD cell subpopulations, equivalent in number to proportions in the same inoculum, were prepared by negative selection. Unfractionated TD cells transferred polyarthritis without in vitro stimulation or conditioning of recipient animals. Abrogation of arthritogenicity by depletion of alpha/beta TCR(+) cells showed that the polyarthritis was transferred by T cells. Negatively selected CD4(+) but not CD8(+) TD cells transferred AA. An arthritogenic subpopulation of CD4(+) T cells, enriched by either negative or positive selection, expressed the activation markers CD25 (IL-2 receptor alpha), CD71 (transferrin receptor), CD134 (OX40 antigen) and MHC class II. Cells expressing these markers were more numerous in TD lymph from arthritic rats than in lymph from normal rats and they included the majority of large CD4(+) T cells. Thus, arthritogenic effector T cells bearing activation markers are released into the central efferent lymph in the late prodrome of AA. Recruitment of these arthritogenic cells to synovium probably determines the polyarticular pattern of AA.

Figures

Fig. 1
Fig. 1
Induction of polyarthritis in DA rats by adoptive transfer of thoracic duct (TD) lymphocytes from arthritic donors. Lymphocytes used for adoptive transfer were pooled from TD lymph collected from groups of four donor rats cannulated 9 days after subcutaneous injection with either CFA (arthritic donors) or PBS (normal donors). Normal syngeneic recipients were injected in a tail vein with the equivalent of the average overnight output (approximately 3 × 108 TD lymphocytes) of cells from one donor. These cells were drawn from either arthritic (n = 4) or normal (n = 4) donor pools. The course of adjuvant-induced arthritis (AA) in DA rats injected at day 0 with complete Freund's adjuvant (CFA) (n = 6) is shown for comparison. Rats receiving CFA were euthanased on or before day 14, in accordance with ethical agreements on disease severity. Joint scores are shown as the mean ± s.d. •, Arthritic donor thoracic duct lymphocytes; ○, normal donor thoracic duct lymphocytes; ×, adjuvant-induced arthritis.
Fig. 2
Fig. 2
(a) Depletion of α/β TCR+ cells abrogates adoptive transfer of polyarthritis by thoracic duct (TD) cells. Pooled TD lymphocytes from arthritic donors were depleted of α/β TCR+ T cells by the use of immunomagnetic beads and a MoAb against the α/β-TCR (n = 4) prior to transfer. A separate aliquot of cells was subjected to the same procedure, except for the use of the isotype-matched control MoAb (n = 4). Joint scores (mean ± s.e.m.) represent the combined results from two separate experiments. •, Control MoAb depleted; □, α/β TCR depleted. (b) Induction of polyarthritis by adoptive transfer of CD4+ T lymphocytes. CD4+ T cells were purified by negative selection from pooled thoracic duct (TD) lymphocytes from arthritic donor rats and injected intravenously into normal syngeneic recipients at the following doses; a1 × 106 (n = 2); a5 × 106 (n = 4); a1 × 107 (n = 6); b5 × 107 (n = 8); b1 × 108 (n = 20). ♦, 1×108 CD4+ T cells; ▪, 5×107 CD4+ T cells; ×, 1×107 CD4+ T cells; ◊, 5×106 CD4+ T cells; ○, 1×106 CD4+ cells. For each group, joint scores are shown as the mean ± s.e.m. Data are combined from 18 separate experiments. Comparisons between transfers of different doses of cells were made using mixed model analysis (see Materials and methods). The course of polyarthritis was significantly different only for those doses (a, b) not bearing a common letter (P < 0·001) (as shown above).
Fig. 3
Fig. 3
The surface antigen phenotype of purified thoracic duct (TD) CD4+ T cells and of the same cells after a secondary depletion of cells expressing the activation markers MHCII, CD25, CD71 and CD134 using immunomagnetic beads and a cocktail of the MoAbs against these markers. The cells were analysed by flow cytometry and the fluorescent intensity is shown plotted against forward light scatter, an index of cell size. TD lymphocytes pooled from four arthritic donor rats were depleted of B cells and CD8+ cells (see Materials and methods). The purified CD4+ T cells were stained with (a) negative control MoAb 1B5 or (b) a mixture of MoAbs against MHC II, CD25, CD71 and CD134. (c) Cells remaining after depletion of those cells expressing the above activation markers (see Materials and methods). The remaining cells were re-stained with the mixture of depleting antibodies. (D). Flow cytometric analysis of CD4+ T cells selected positively on the basis of their activated phenotype. CD4+ T cells were prepared by negative selection (see Materials and methods) from TD lymphocytes pooled from four arthritic donor rats. They were then stained with a mixture of MoAbs against MHCII, CD25 and CD71. After positive selection of the labelled cells using a Dynal rabbit antimouse IgG1 CELLection kit (see Materials and methods), they were re-stained with the same mixture of MoAbs.
Fig. 4
Fig. 4
Depletion of an activated population abrogates adoptive transfer of polyarthritis by thoracic duct (TD) CD4+ T cells. Purified CD4+ T lymphocytes were prepared from pools of TD lymph from arthritic donor rats by negative selection. They were further depleted of cells expressing activation markers and transferred by intravenous injection to normal syngeneic recipients. MoAbs directed against activation markers were used either singly or in combination or were replaced by the isotype-matched control MoAb (see below). Equal numbers of cells remaining after the secondary depletion with immunomagnetic beads were transferred to recipients in each experiment. The number of cells transferred represented approximately the yield obtained by each procedure from the overnight output of one donor rat and ranged from 5 × 107 to 1 × 108. (a) Transfers of CD4+ T lymphocytes remaining after depletion with a control MoAb (n = 6) or a mixture of MoAbs OX6, OX39, OX26 and OX40 against MHCII, CD25, CD71 and CD134, respectively (n = 5). Joint scores (mean ± SEM) represent the results from three separate experiments. Mixed model analysis showed the curves to be significantly different for days 5–17 (P < 0·0001). •, Control MoAb depleted; ○, anti-MHCII + anti-CD25+ anti-CD71 + anti-CD134 depleted. (b) Transfers of CD4+ T lymphocytes remaining after depletion with either control MoAb IB5(n = 19), MoAb OX39 against CD25 (n = 8), MoAb OX6 against MHC II (n = 12) or a mixture of the MoAbs against MHCII and CD25 (n = 4). •, Control MoAb depleted; ◊, anti-CD25 depleted; ×, anti-MHCII depleted; □, anti-MHCII + anti-CD25 depleted. Data are combined from 10 separate experiments. Comparisons between transfers of cells from each depletion protocol were made using mixed model analysis (see Materials and methods). The course of polyarthritis was significantly different (P < 0·0001) only for the preparations and periods not bearing a common letter (a, b, c) as designated in the following; •, depletion with negative control MoAb, adays 2–30; ◊, depletion of CD25+ cells, bdays 2–5, 20–30, cdays 6–19; ×, depletion of MHC class II+ cells, bdays 2–30; □, depletion of cells expressing CD25 and/or MHC class II,b days 2–30.
Fig. 5
Fig. 5
Adoptive transfer of polyarthritis by CD4+ T cells purified from TD lymph by positive selection using MoAbs against MHC II, CD25 and CD71. (a) CD4+ T cells were purified by negative selection from TD lymph, pooled from four arthritic donor rats and cells expressing one or more of the activation markers MHCII, CD25 and CD71 were selected positively using MoAbs OX6, OX39 and OX26, respectively (see Materials and methods). The positively selected (1 × 107 cells per rat, n = 2) or the residual (3 × 108 cells per rat, n = 2) fractions were transferred by intravenous injection to normal syngeneic rats. The proportions of cells expressing the activation markers in the fractions were 97% (see Fig. 3d) and 14% (not shown), respectively. Joint scores are shown as the mean ± s.d. (n = 2). ▪, 1×107 CD4+ T cells positively selected with anti-MHCII + anti-CD25 + anti-CD71; ▾, 3×108 residual CD4+ T cells. In a separate experiment (b) and (c), a comparison of potency was made between the complete CD4+ population of T cells from pooled TD lymph from arthritic donor rats and the activated subset purified from these cells by positive selection of cells expressing MHC II, CD25 and CD71. (b) Transfer of positively selected cells at doses of 1 × 106 and 1 × 107 cells per recipient. ▴, 1×107 CD4+ T cells positively selected with anti-MHCII + anti-CD25 + anti-CD71; ▪, 1×106 CD4+ T cells positively selected with anti-MHCII + anti-CD25 + anti-CD71; (c) Transfer of the CD4+ T cells (but without selection for activation marker-positive cells) at doses of 1 × 107 and 5 × 107 cells per rat. ▵, 5×107 CD4+ T cells; ○, 1×107 CD4+ T cells. Joint scores are shown as the mean ± s.d. (n = 2). Note that although the results using CD4+ T cells and the activated subset of CD4+ T cells are shown on separate panels, the cells were both derived from same donor pool.

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