Effector CD4 cells are tolerized upon exposure to parenchymal self-antigen

Abstract

It has long been established that exposure of naive T cells to specific Ag in the absence of adjuvant leads to tolerization. Nonetheless, the potential of effector CD4 cells to be tolerized has been less well characterized. To address this issue, we have used an adoptive transfer system in which naive TCR transgenic hemagglutinin (HA)-specific CD4(+) T cells are initially primed to express effector function upon exposure to an immunogenic recombinant vaccinia virus expressing HA, and then exposed to forms of HA that are tolerogenic for naive CD4 cells. HA-specific effector CD4 cells residing in both the spleen as well as in two separate nonlymphoid tissues were tolerized upon exposure to high doses of exogenous soluble HA peptide. Additionally, tolerance could also be induced by bone marrow-derived APCs that cross-present parenchymally derived self-HA. Thus, effector CD4 cells are susceptible to similar tolerogenic stimuli as are naive CD4 cells.

FIGURE 1

Simultaneous exposure of naive CD4 cells to virally derived and self cognate Ag leads to impaired virally induced effector function. CFSE-labeled naive clonotypic CD4 cells were adoptively transferred into the following recipients: NT + vacc-HA (vacc-HA-infected NT mice), C3-HA (noninfected C3-HA^low transgenic mice), and C3-HA + vacc-HA (vacc-HA-infected C3-HA^low transgenic mice). Six days later, the clonotypic CD4 cells were recovered from spleens to determine their proliferative responses and abilities to express intracellular IFN-γ following in vitro restimulation. A, Representative FACS histogram plots of CFSE dilution (left panels; with a dotted reference line placed immediately to the left of the undivided cells) and intracellular IFN-γ expression (right panels; the percentage of cytokine-positive cells and the level of cytokine expression (expressed as the mean fluorescence (MF)) are shown above and below the reference bar, respectively). B, The frequencies of clonotypic CD4 cells. C, Total intracellular IFN-γ expression was calculated as the product of the percentage of IFN-γ-expressing clonotypic CD4 cells and the level of IFN-γ expression (MF) on these positive cells, and is expressed in arbitrary units, as previously described (26). n = 3–5 for each group, and the data shown are representative of two separate experiments.

FIGURE 2

In vivo treatment of effector CD4 cells with soluble peptide leads to an impaired ability to produce IFN-γ and IL-2 as well as to undergo blastogenesis following Ag restimulation. Vacc-HA-infected and non-infected NT adoptive transfer recipients of naive CFSE-labeled clonotypic CD4 cells were treated with 280 µg of soluble HA peptide as indicated, and the clonotypic CD4 cells were recovered from spleens on either day 7 or 14 for analysis (refer to legend). A, Total intracellular cytokine expression following in vitro restimulation. B, The frequencies of clonotypic CD4 cells corresponding to A and C. C, Blastogenesis of clonotypic CD4 cells. Representative FACS histogram plots of forward scatter (FSC) on gated clonotypic CD4 cells are shown on the left, with the percentage of blasting cells indicated above the reference bar. The graph on the right shows the quantitative analysis, with the experimental groups that received a peptide bolus 20 h before recovery represented with hatched bars. For A–C, n = 3–6 for each group, and the data were pooled from three separate experiments that each contained multiple treatment groups to verify consistency. D, CFSE-dilution histogram plots for naive clonotypic CD4 cells adoptively transferred into NT recipients that had been treated with 280 µg soluble HA peptide at the indicated times before transfer. Analysis was performed on spleens 3 days posttransfer. The percentage of clonotypic CD4 cells (Thy-1.1⁺ and 6.5⁺) in the initial population that did not divide (shown in the upper right corner) was calculated as previously described (52).

FIGURE 3

In vivo treatment of Th1 effector CD4 cells with soluble peptide does not induce conversion into Th2 effector or regulatory CD4 cells. Vacc-HA-infected NT adoptive transfer recipients of CFSE-labeled naive clonotypic CD4 cells were treated with or without 280 µg of soluble HA peptide on days 6 and 10, and analysis of spleens was performed on day 14. A, Total intracellular cytokine expression following in vitro re-stimulation (n = 3 for both groups). B, Representative FACS histogram plots of CD25 expression on primed clonotypic CD4 cells (vacc-HA) and peptide-treated counterparts (vacc-HA + P), with the percentage of positively expressing cells shown above the reference bar. CD25 expression on naive and blasting (i.e., activated) clonotypic CD4 cells is shown for comparison. Note that the background staining on naive clonotypic CD4 cells is slightly elevated due to a compensation effect resulting from high CFSE fluorescence.

FIGURE 4

Effector CD4 cells residing in non-lymphoid tissues are tolerizable. Vacc-HA-infected NT adoptive transfer recipients of CFSE-labeled naive clonotypic CD4 cells were treated with or without 280 µg of soluble HA peptide on days 6 and 10. On day 14, animals were perfused, and lymphocytes were prepared from the spleen, liver, and lung for analysis. A, Total intracellular cytokine expression following in vitro restimulation. n = 3 for all groups, except for the liver + peptide group, in which n = 2 because there was an insufficient number of clonotypic CD4 cells extracted from one of the samples for analysis. B, The frequencies of clonotypic CD4 cells corresponding to A.

FIGURE 5

Effector CD4 cells undergo tolerization upon encountering parenchymally derived self-Ag. Day 6 clonotypic effector CD4 cells recovered from vacc-HA-primed primary adoptive transfer recipients were relabeled with CFSE and retransferred into the indicated secondary recipients. Eight days later, they were recovered from spleens for analysis. A, Representative FACS histogram plots showing CFSE-dilution profiles with a dashed reference line placed immediately to the left of the undivided cells. B, Frequencies of clonotypic CD4 cells corresponding to A, C, and D. C, Intracellular cytokine expression of clonotypic CD4 cells following in vitro restimulation. Representative histogram plots of intracellular IFN-γ expression in the different secondary recipient groups, as well as in the primary effectors immediately before the retransfer (1°) are shown on the left. Total intracellular IFN-γ and IL-2 expression is shown on the right. D, Blastogenesis of clonotypic CD4 cells following recovery from secondary recipients. Clonotypic effectors given a single peptide bolus on day 6 and analyzed on day 7 (data taken from Fig. 2C) are shown as a reference (C). n = 5–8 for each group, and the data were pooled from three separate experiments that each contained multiple treatment groups to verify consistency.