Abstract
CD8 T cells can play both a protective and pathogenic role in inflammation and autoimmune development. Recent studies have highlighted the ability of CD8 T cells to function as T follicular helper (Tfh) cells in the germinal center in the context of infection. However, whether this phenomenon occurs in autoimmunity and contributes to autoimmune pathogenesis is largely unexplored. In this study, we show that CD8 T cells acquire a CD4 Tfh profile in the absence of functional regulatory T cells in both the IL-2–deficient and scurfy mouse models. Depletion of CD8 T cells mitigates autoimmune pathogenesis in IL-2–deficient mice. CD8 T cells express the B cell follicle–localizing chemokine receptor CXCR5, a principal Tfh transcription factor Bcl6, and the Tfh effector cytokine IL-21. CD8 T cells localize to the B cell follicle, express B cell costimulatory proteins, and promote B cell differentiation and Ab isotype class switching. These data reveal a novel contribution of autoreactive CD8 T cells to autoimmune disease, in part, through CD4 follicular-like differentiation and functionality.
This article is featured in In This Issue, p.3
Introduction
The development of autoimmunity involves both a breakdown in tolerance control mechanisms and complex interactions between immune cells. Some of these cells promote disease, whereas others act to block this dysregulation. As disease progresses, immune activation is amplified, self-perpetuating the lymphoproliferation, inflammation, and self-destruction associated with autoimmunity. The process initiated by inflammatory and Ag signaling promotes T cell proliferation, differentiation, and acquisition of T cell effector functions. Autoimmunity in the IL-2–deficient (knockout [KO]) mouse model results from reduced T regulatory cell (Treg) frequency and functionality (1–3), promoting dysregulation of the T effector response. CD4 Th1 cells subsequently promote the production of anti-RBC IgG Abs and bone marrow failure dependent on IFN-γ secretion (4–6). Although there is a clearly established role for CD4 T cells in promoting Ab-mediated disease, the importance of CD8 T cells in these diseases has been less explored.
B cell responses to self-antigens during autoimmune disease are induced and enhanced by germinal center (GC) reactions in the peripheral lymphoid organs (7, 8). Within the GC, activated, Ag-specific B cells undergo clonal expansion, BCR somatic mutation, affinity maturation and Ab class switching, and differentiate into memory and long-lived plasma cells. GC reactions begin at the border of the B cell follicle and the T cell zone, where CD4 T follicular helper (Tfh) cells interact with B cells (9). CXCR5 upregulation and CCR7 downregulation facilitates migration of CD4 Tfh and activated B cells into the follicle. Engagement of several interactions between the activated CD4 Tfh and B cells (including ICOS-ICOSL and CD40-CD40L) ensures optimal GC reactions and CD4 Tfh cell development by promoting the transcription factor, Bcl6 (10). CD4 Tfh cells are required for the production of high avidity, class-switched Abs (11).
Abnormal activation of CD4 Tfh cells, or loss of regulation, can promote Ab-mediated autoimmune disease (12–14). Follicular CD4 Tregs are essential inhibitors of GC interactions by mediating T cell help to B cells (7). In the absence of CD4 follicular Tregs, CD4 Tfh cell expansion results in autoantibody generation and autoimmune disease (13, 15). Similarly, CD8 Tregs also control self-reactive cells, and their elimination exacerbates autoimmune disease (16, 17). Like CD4 T cells, CD8 T cells differentiate into effector subsets based upon their transcription factor expression and cytokine production, and these cytokines may amplify CD4 T cell responses or act through mechanisms unique to CD8 T effectors (17). However, regulation and function of distinct CD8 T cell subsets are less clearly delineated as compared with CD4 helper cells.
CD8 T cell effectors located within the GC have recently been described (18–22). In rheumatoid arthritis synovial ectopic follicles, CD8 T cells make up the majority of the infiltrating T cells and express CD40L, which is important in B cell GC reactions (19). Furthermore, the CD8 T cells are required for the formation and maintenance of ectopic GCs (18). CD8 T cells expressing CXCR5 also develop in chronic viral infection and under inflammatory conditions (20, 21, 23–25). CXCR5+ CD8 T cells are localized in the B cell follicle in human tonsil, and these cells support B cell survival in ex vivo culture (26). CXCR5+ CD8 memory T cells within the GC control viral load in lymphocytic choriomeningitis virus and SIV infections and express many of the genes associated with CD4 Tfh cell differentiation and function (20, 23). Together, these data suggest that under specific disease conditions, CD8 T cells may acquire unique functionality within the GC. Whether CD8 T cells function in GC reactions in autoimmune disease is largely unexplored.
Materials and Methods
Mice, immunizations, and Ab depletions
BALB/c IL-2–KO mice, wild-type (WT), and IL-2–heterozygous (HET) littermate controls (IL-2–HET and WT; referred to as WT) were used. IL-2–KO autoimmune disease is not gender specific, only age is used to determine data inclusion, as specified in each figure legend. BALB/c hemizygous male Foxp3sf/Y (scurfy) mice and HET female FoxPsf/+ (scurfy-HET) mice were purchased from The Jackson Laboratory (27). Both gender and age are used to determine data inclusion in scurfy disease. Our breeding setup is restricted to scurfy male mice, and age-matched female scurfy-HET littermates were used as controls where indicated. For immunization, mice were treated by i.p. injection with 100–200 μg keyhole limpet hemocyanin (KLH) in CFA at −15 to −26 d, followed by a second i.p. injection at −5 d, as previously described (7, 8). CD4 or CD8 depletions were performed by i.p. injection of 20 μg anti-CD4 (GK1.5) or anti-CD8 (2.43) Ab per gram weight three times per week, from day 8 to 16. IL-2 depletions were performed by i.p. injection of 20 μg anti–IL-2 (JES6-1A12) Ab per gram weight three times per week between days 7 and 15. Abs were purchased from the University of California (UC), San Francisco mAb Core or Bio X Cell. All mice were bred and maintained in our specific pathogen-free facility in accordance with the guidelines of the Department of Animal Research Services at UC Merced. The UC Merced Institutional Animal Care and Use Committee approved all animal procedures.
Complete blood counts
Cardiac punctures or eye bleeds were performed immediately following cervical dislocation, and blood was collected in heparinized tubes (28). Complete blood counts were evaluated within 24 h on a Hemavet 950 Veterinary Hematology System.
Microscopy and immunofluorescence
+ GCs were traced in ImageJ using the freehand selection tool, and the area was determined. CD8+ cells within the GC were marked using the multipoint selection tool when a dark center (nucleus) surrounded by CD8 surface staining could be identified. GC CD8 T cells within the GC area were quantified manually.
Flow cytometry and cell sorting
Prior to cell sorting, pooled splenocytes and lymphocytes were depleted of non–T cells using EasySep Mouse PE Selection Kit according to the manufacturer’s instructions (STEMCELL Technologies) to remove B220-PE+, CD11c-PE+, CD11b-PE+, and Gr-1-PE+ cells. CD4 Tfh (CXCR5+PD-1hi) and CD8 Tfc cells (CXCR5+PD-1hi) were sorted from IL-2–KO mice, CD4 Tfh cells (CXCR5+PD-1hi) were sorted from KLH-immunized mice, and bulk CD4 and CD8 T cells were sorted from WT or IL-2–KO mice, as indicated. B cells (CD19+TCRβ−CD11c−CD11b−Gr-1−) were sorted from pooled WT spleens. All sorts were performed with >90% purity on the Aria II cell sorter (BD Biosciences).
RBC Ab detection
Serum RBC Ab levels were detected, as previously described (30). Freshly isolated RBCs were washed three times in PBS and resuspended to 1% RBCs. Ten microliters of 1% RBCs were incubated with anti-mouse IgM-FITC (1:150; on ice) or anti-mouse IgG-FITC (1:50; at 37°C; Jackson ImmunoResearch Laboratories). The percentage of RBCs bound by Ab was determined by flow cytometry.
T cell stimulations
Harvested cells were stimulated at 37°C with 50 ng/ml PMA and 500 ng/ml ionomycin for 5 h with Brefeldin A or monensin added during the final 4 h. IL-2 cytokine production was determined by intracellular flow cytometry. Anti-CD40L (MR1) was added directly to cells during the stimulation, as previously described (31). Cells were stained poststimulation for CD4 Tfh and CD8 Tfc cell markers (CXCR5+PD-1hi2 goat anti-human IgG (Jackson ImmunoResearch Laboratories) at 4°C (32).
In vitro T cell and B cell culture assays
T and B cell stimulation was performed, as previously described (22). Indicated T cell populations were plated at 5 × 1044 sorted WT B cells per well and with 1 μg/ml anti-CD40 (IC10) and 5 μg/ml F(ab′)2 goat anti-mouse IgMμ (Jackson ImmunoResearch Laboratories) for 6 d. B cell supernatant was analyzed for Ab production by ELISA.
T cell adoptive transfer assays
IL-2–KO T cells were adoptively transferred, as previously described (4). 1 × 106 CD4 T cells, 2 × 106 CD8 T cells, or a combination of each were transferred into TCRα-KO mice via eye injection. Two days after cell transfer, mice were immunized i.p with 200 μg of KLH in CFA. Seven days after cell transfer, mice were reimmunized i.p. with 100 μg of 4-Hydroxy-3-nitrophenylacetyl (NP)–conjugated KLH in IFA.
Ab ELISA
Total IgG was determined by ELISA, as described (28). Serum and culture supernatant samples were prepared in PBS/1% BSA. Serum from depletion experiments was prepared at 1:50,000 dilution, a standard curve of purified mouse IgG (SouthernBiotech), stimulated B cell supernatants were prepared at 1:50 dilution, and serum from adoptive transfer experiments was diluted to 1:10,000 dilution for IgG2a and IgG2b or 1:100,000 dilution for IgG1. Abs were detected with HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, IgG2b, or IgG3 (SouthernBiotech), then developed with TMB Peroxidase Substrate (Vector Laboratories) following manufacturers’ instructions. Plates were stopped with 1 N sulfuric acid and read on a Victor 3 1420 Multilabel Counter plate reader (PerkinElmer) at 450 nm. When applicable, a standard curve of purified mouse IgG (SouthernBiotech) was used to interpolate IgG concentrations from a sigmoidal standard protein curve.
RNA isolation and analysis of RNA next-generation sequencing
CD8 cells were sorted from 12-d-old IL-2–KO and WT mice to ≥85% purity. Samples were quick frozen and shipped to Expression Analysis for total RNA isolation using Illumina TrueSeq Stranded Total RNA Sample Preparation Kit. Eight samples were sequenced, four biological replicates each for IL-2–KO and WT mice, producing 2 × 50 paired-end reads using the Illumina HiSeq 2500 platform. Raw reads were provided by Expression Analysis and were used for further analyses. Adapter removal and quality trimming at the Q20 level were performed using Atropos v1.1.17 (33) with Python v3.6.2. Read pairs were removed if either read was <20 bp trimming. Rsubread v1.28.1 (34) was used to perform read alignment, reporting up to 10 equally likely mapping locations. Read pairs that could not be aligned together were aligned individually. The genome used for alignment was C57BL/6J of GRCm38/mm10 (GCF_000001635.20). Read summarization was performed on the gene level using featureCounts (35) using annotations from a modified version of the annotations for GRCm38/mm10 containing only protein coding genes. Multimapping reads were treated as fractional counts when mapping to several genes, mapping across more than one gene, and read pairs in which ends mapped to different chromosomes were discarded. Genes that had <1 count per million in three to four samples were discarded. Remaining gene counts were normalized using trimmed mean of M-values (36). Differential expression analysis was performed using limma v3.34.9 (37) with voom-transformed read counts (38). Genes were considered differentially expressed if their p value was <0.05 after the false discovery rate was controlled (39). Read mapping, summarization, and differential expression analysis were performed using R v3.4.3. Differentially expressed genes were annotated with their biological process Gene Ontology group using Panther 13.1 (40) and the Gene Ontology database released on February 2, 2018. Sequence data were uploaded to National Center for Biotechnology Information GEO under accession number GSE112540 at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE112540.
Real-time PCR
Total RNA was isolated from cells using an RNeasy kit (Qiagen), and cDNA syntheses were conducted according to the manufacturer’s instructions with Superscript III First-Strand Synthesis SuperMix for RT-PCR (Invitrogen). Real-time PCR analysis was conducted in duplicates using Mx3000P (Stratagene California) with Fast SYBR Green RT-PCR Master Mix (Bio-Rad). Averages of the collected data were normalized to β-actin or HPRT. Relative expressions (ΔΔCt) were calculated to the indicated cell population.
Statistics
GraphPad (Prism) was used for statistics. Differences between two experimental groups were determined by unpaired Student t test unless otherwise noted. A Mantel–Cox log-rank test was used to analyze Kaplan–Meier survival curves. Protein concentrations from a standard protein curve were interpolated using a sigmoidal four parameter logistic standard curve with x as log(concentration). Statistically significant differences in adoptive transfer and supernatant culture experiments were determined by one-way ANOVA with Bonferroni correction.
Results
Depletion of either CD4 or CD8 T cells reduces anemia and anti-RBC Abs and prolongs survival
We have previously demonstrated that early death in IL-2–KO mice is driven in part by autoimmune hemolytic anemia that requires IFN-γ and is CD4 Th1–mediated (5, 6). We found that CD8 T cell numbers were increased on average by 3-fold in the LNs and spleen of IL-2–KO mice (Supplemental Fig. 1A). Both splenic and LN CD8 T cells expressed decreased CD62L and elevated CD44 and CD69, indicating an activated state (Supplemental Fig. 1B, 1C). CD8 T cells have been shown to promote and inhibit autoimmunity in multiple models due to both regulatory and effector responses (4, 8, 19, 41). As the population of IL-2–KO CD8 T cells was activated and expanded, we next determined how and to what extent CD8 T cells contributed to autoimmunity in IL-2–KO mice.
To evaluate the contribution of CD8 T cells to autoimmune disease, we eliminated CD8 T cells or CD4 T cells as a control, prior to disease onset, by treating with anti-CD4– or anti-CD8–depleting Abs (Fig. 1A). As expected, depletion of CD4 T cells significantly delayed disease onset with a median survival of 14 wk, as compared with 19–25 d survival for PBS-treated IL-2–KO mice (Fig. 1B). Surprisingly, depletion of CD8 T cells also significantly prolonged survival, with a median survival of 12 wk. In concordance with augmented survival, IL-2–KO mice with depleted CD4 or CD8 T cells had increased hemoglobin levels relative to PBS-treated IL-2–KO mice (Fig. 1C). Both T cell depletions also significantly reduced B cell numbers (Fig. 1D) and frequency of IgM and IgG autoantibodies bound to RBCs (Fig. 1E, 1F) relative to PBS-treated IL-2–KO mice. In the absence of CD8 T cells, serum IgG1 (but not IgG3, IgG2b, or IgG2a) was significantly reduced in comparison with PBS-treated IL-2–KO mice (Fig. 1G). Although these data confirm that CD4 T cells play a critical role in autoimmune progression, they also reveal the contribution of CD8 T cells to the rapid autoimmunity that occurs in IL-2–KO mice. Furthermore, these data demonstrate that CD8 T cells facilitate enhanced B cell expansion and Ab production in IL-2–KO mice, as these outcomes are significantly reduced when CD8 T cells are eliminated.
Depletion of IL-2–KO CD4 or CD8 T cells prolongs survival and delays Ab production. (A) Schematic describing the Ab depletion. Peripheral blood and LN cells were isolated at 18–21 d of age. (B) Kaplan–Meier survival plots. Statistics were performed to test differences relative to untreated IL-2–KO mice. (C) Hemoglobin levels were measured from peripheral blood by complete blood count (CBC) at day 19 of age. (D) Total LN B220+ B cell numbers were determined by flow cytometry and cell counting. (E and F) RBCs were stained with anti-mouse IgM-FITC (E) or IgG-FITC (F) and analyzed by flow cytometry to detect the percentage of RBC bound by Abs. (G) Serum IgG1, IgG3, IgG2b, and IgG2a levels were determined by ELISA. (C–G) Each symbol indicates an individual animal. Statistics tests were performed relative to IL-2–KO and unpaired Student t test with a Welch correction. *p < 0.05, **p < 0.01, ***p < 0.001.
Transcriptional profiling of CD8 T cells during early autoimmunity
To determine mechanistically how CD8 T cells contribute to Ab-mediated autoimmune development in the absence of functional Tregs, we examined early gene dysregulation in IL-2–KO CD8 T cells. We performed RNA sequencing of CD8 T cells sorted from 12-d-old mice, as this is the earliest time point at which IL-2–KO CD8 T cells have been shown to be activated (42). Bulk CD8 T cells were sorted from the peripheral LNs of four sets of pooled IL-2–KO and littermate control WT mice. Differential expression analysis identified 2226 genes (1290 upregulated; 936 downregulated) that showed significant (p < 0.05) differences in expression in IL-2–KO CD8 T cells relative to WT CD8 T cells (Supplemental Table I). Differentially expressed genes were grouped into several biological process Gene Ontology categories, including metabolic processes, cellular processes, and biological regulation (Fig. 2A). Several genes upregulated in IL-2–KO CD8 T cells were genes involved in cytolytic function (granzymes, tbx21, fasl), but we also identified differential expression of costimulatory molecules and follicular helper-associated genes such as icos, cd28, il21, and bcl6 (Fig. 2B). Further evaluation of RNA sequencing data confirmed the profiles of cytolytic gene expression in individual IL-2–KO mice (Fig. 2C). Based on the observed reduction in B cell numbers and Ab production in the absence of CD8 T cells (Fig. 1), we focused our analysis on the expression of costimulatory molecules and genes involved in B cells. This analysis revealed a profile of gene expression in CD8 T cells comparable to that described in CD4 Tfh cells (Fig. 2D). IL-2–KO CD8 T cells expressed elevated cxcr5, sh2d1a (SAP), icos, bcl6, il21, and several other genes that define CD4 Tfh cells, as further confirmed by real-time PCR (Fig. 2E). Thus, during early autoimmune initiation, CD8 T cells acquire a gene expression profile associated with varying functional roles, including a CD4 Tfh cell–like role.
Cytolytic and follicular helper profile of IL-2–KO CD8 T cells. RNA sequencing of four independent WT and IL-2–KO CD8 T cell samples pooled from LN of 12-d-old mice. (A) Gene Ontology analysis of all 2226 differentially expressed genes in IL-2–KO CD8 T cells relative to WT. (B) Volcano plots displaying log2 fold change gene expression of IL-2–KO relative to WT CD8 T cells versus log10 p value. Select differentially expressed genes are labeled in the plot. Data are organized by color to indicate both log fold change (LFC) and p value. (C and D) Heat maps showing CD8 T cell expression data. Color indicates gene expression by Z-score, and * indicates IL-2–KO gene expression with statistical significance relative to WT. Differential expression of select cytolytic-associated genes (C) and CD4 Tfh-associated genes (D). (E) mRNA expression of select genes in two independent experiments from 12-d-old IL-2–KO CD8 T cells relative to WT CD8 T cells. Dashed line indicates a fold change of 1, and error bars indicate SD.
CD8 Tfc cells develop during systemic autoimmune disease
We next evaluated protein expression of CD4 Tfh cell–associated genes in IL-2–KO CD8 T cells. In naive mice, a small population (0.1–0.5%) of CXCR5+PD-1hi CD4 Tfh cells has been described (43, 44). We used this population and percentage range, in addition to fluorescence-minus-one controls, to confirm our gating strategy for CD8 T cells. Using these stringent conditions, a very small population of ≤0.2% CD8 T cells expressed CXCR5 and PD-1 in naive WT mice. In contrast, the same markers were significantly expanded among both CD4 and CD8 T cells in IL-2–KO LN (7.0 ± 1.8% of CD4 T cells and 2.5 ± 1.2% of CD8 T cells; Fig. 3A). Similarly, CXCR5+PD-1hi CD4 and CXCR5+PD-1hi CD8 T cells significantly increased in frequency and total number in the spleens of IL-2–KO mice relative to naive WT (Supplemental Fig. 2A). To confirm select gene expression patterns identified in the bulk CD8 T cell RNA sequencing, we performed real-time PCR analysis of IL-2–KO CD4 Tfh and CD8 Tfc cells relative to IL-2–KO CD4 non-Tfh cells and CD8 non-Tfc cells, respectively. CD8 Tfc cell mRNA levels of bcl6, icos, cd28, sh2d1a, il6ra, and ccr7 were comparable to CD4 Tfh cells (Fig. 3B). Thus, based on their surface phenotype and gene expression profile, we defined the CXCR5+PD-1hi CD8 T cells as CD8 Tfc cells.
CD8 T cells express markers of follicular helpers. (A) Flow cytometric analysis of CXCR5 and PD-1 expression on CD4 or CD8 T cells from 18- to 21-d-old IL-2–KO or WT LN gated on B220−CD11c−CD11b−GR-1−. Representative flow plots, frequency, and total number of CXCR5+PD-1hi CD4 and CD8 T cells are shown. (B) Real-time PCR comparing relative gene expression in IL-2–KO CD4 Tfh or CD8 Tfc cells (CXCR5+PD-1hi) with IL-2–KO CD4 CXCR5−PD-1lo (non-Tfh) or CD8 CXCR5−PD-1lo (non-Tfc) cells, respectively. Dashed line indicates fold change of 1, and error bars indicate SD. (C) Percent and total number of CD4 Tfh and CD8 Tfc cells in IL-2–KO and WT mice from 12 to 20 d of age. (D and E) Representative flow plots and MFI quantification of surface expression of ICOS (D) and Bcl6 (E) in WT naive bulk CD4 and CD8 T cells, IL-2–KO CD4 non-Tfh or CD8 non-Tfc cells, and IL-2–KO CD4 Tfh and CD8 Tfc cells. (A, D, and E) Each symbol indicates an individual animal. (A and C–E) Data are representative of three to six independent experiments. (B) Data are representative of two independent experiments. Statistics: unpaired Student t test relative to WT with a Welch correction. *p < 0.05, **p < 0.01, ***p ≤ 0.001.
A detectable population of CD8 Tfc cells was identified in IL-2–KO mice at day 12 by flow cytometry and continued to expand in both frequency and total number over time (Fig. 3C). Although, CXCR5+PD-1hi T cells comprise only a small fraction of the expanded CD4 and CD8 T cell population observed in IL-2–KO mice (Supplemental Fig. 2B). We next evaluated CD8 Tfc cells for the expression of other proteins known to be involved in B cell interactions within the follicle. ICOS is highly expressed during CD4 Tfh cell differentiation and promotes the expression of Bcl6, a master regulator of CD4 Tfh cell fate that promotes CD4 Tfh/B cell interactions (45, 46). IL-2–KO CD8 Tfc cells expressed increased ICOS and Bcl6, similar to IL-2–KO CD4 Tfh cells, in comparison with both naive WT CD8 T cells and IL-2–KO CD8 non-Tfc cells (Fig. 3D, 3E, Supplemental Fig. 2C, 2D). Together, mRNA and surface expression of effector proteins that typically describe CD4 Tfh cells confirm the identity of CD8 Tfc cells in systemic autoimmunity.
Next, we validated that CD8 Tfc cells develop under IL-2–sufficient autoimmune conditions (47, 48). Scurfy mice lack functional Treg development, resulting in early systemic autoimmune disease (48). We first confirmed that, despite a reduction in the total number of IL-2–producing CD4 and CD8 T cells in the scurfy mice, the frequency of IL-2–producing cells was comparable to WT mice (Fig. 4A) (47). We identified a significantly expanded population of CXCR5+PD-1hi CD4 Tfh cells (3.0 ± 0.6%) and CXCR5+PD-1hi CD8 Tfc cells (1.0 ± 0.3%) in scurfy LNs compared with scurfy-HET LNs (Fig. 4B). Depleting IL-2 in scurfy mice using a neutralizing Ab or by genetic cross to the IL-2–KO background resulted in a higher percentage of CD4 Tfh and CD8 Tfc cells as compared with scurfy mutants but not IL-2–KO mice (data not shown). Similar to IL-2–KO mice, CD8 Tfc cells in scurfy mice expressed ICOS and Bcl6 (Fig. 4C, 4D). Together, these data demonstrate even under IL-2–sufficient conditions that CD8 Tfc cells can be identified during autoimmunity induced by the absence of functional Tregs. However, differences in CD8 Tfc cell frequency between the IL-2–KO and scurfy mice suggest that IL-2 may play a role in regulating the expansion of this distinct CD8 T cell population.
Autoimmune scurfy mice develop CD8 Tfc cells. Flow cytometric analysis of scurfy and scurfy-HET littermate control LNs. (A) Quantification of IL-2–producing CD4 and CD8 T cell frequency and total cell number after PMA and ionomycin stimulation. (B) CXCR5 and PD-1 expression on CD4 or CD8 T cells from 18- to 19-d-old scurfy or scurfy-HET gated on live B220−CD11c−CD11b−GR-1−. Representative flow plots, frequency, and total number of CXCR5+PD-1hi CD4 and CD8 T cells are shown. (C and D) Representative flow plots and quantification of MFI of expression of ICOS (C) and Bcl6 (D) in scurfy-HET naive bulk CD4 and CD8 T cells, scurfy CD4 non-Tfh or CD8 non-Tfc cells, and scurfy CD4 Tfh and CD8 Tfc cells (CXCR5+PD-1hi), following stimulation with PMA and ionomycin. Each symbol indicates an individual animal. Data are representative of three to six independent experiments. Statistics: unpaired Student t test relative to scurfy-HET (A, B, and D) with a Welch correction in (C). **p < 0.01, ***p ≤ 0.001.
GC localization of CD8 T cells
CXCR5 expression by B cells and CD4 Tfh cells allows colocalization of these cells into the follicle, providing a site for productive T cell/B cell interactions. During chronic viral infection, CXCR5+CD8 T cells have been reported both within the B cell follicle and, primarily, excluded from the follicle (20, 21, 23). CXCR5+CD8 T cells localize to the follicle in ectopic GCs but not in the spleen and LN during arthritis and influenza infection (18, 22). We investigated whether CD8 T cells enter the B cell follicle during systemic autoimmunity. As IL-2–KO mice develop abnormal GC structure during late-stage disease (42, 49), we selected early disease–stage IL-2–KO mice with normal, albeit large, splenic gross morphology. To determine if CD8 Tfc cells were capable of GC localization, we evaluated GL-7 expression, a known GC-specific marker (50). Both IL-2–KO CD4 Tfh and CD8 Tfc cells express elevated levels of GL-7 (Fig. 5A). To examine CD8 T cell localization within IL-2–KO GCs, we compared immunized WT spleens with IL-2–KO spleens. GCs were defined as GL-7+ GC B cells within IgD+ B cell follicles (Fig. 5B). IL-2–KO spleens had significantly larger GC areas compared with immunized WT spleens. Nonetheless, 3.9-fold more CD8 T cells were present within a comparable area of IL-2–KO GC relative to those observed in immunized WT GCs (Fig. 5C). To determine if CD8 Tfc cells are capable of providing B cell help, we tested for the expression of CD154 (CD40L), as CD40/CD40L interactions are known to be crucial for B cell activation and Ab class switching in normal and autoimmune settings (51, 52). IL-2–KO CD8 Tfc cells were found to express CD40L upon stimulation (Fig. 5D). These results indicate that IL-2–KO CD8 T cells are capable of coexpressing B cell zone-specific markers (CXCR5 and GL-7), localizing to the GC, and expressing helper proteins, which may promote CD8 Tfc and B cell interactions that influence autoimmune disease.
CD8 T cells localize to the GC during autoimmune disease. (A) Representative flow plots and MFI quantification of GL-7 expression in IL-2–KO and WT splenocytes in the indicated populations. (B) Immunofluorescence staining of B cells (red, IgD), GCs (blue, GL-7), and CD8 (green, CD8α) of spleens from 18- to 21-d-old IL-2–KO and KLH-immunized WT (Imm. WT) mice. White dotted lines indicate the GC outline. White scale bar, 100 μm. (C) Quantification of GC area (square micrometer) and CD8 T cells (per square millimeter). (D) Representative flow plots and MFI quantification of CD40L expression in IL-2–KO and WT CD4 and CD8 T cells stimulated with PMA and ionomycin. (A, C, and D) Each symbol indicates an individual animal. (A) Data from four independent experiments. (B and C) Data are representative of 5 spleens and 19 GCs from IL-2–KO mice or 4 spleens and 17 GCs in Imm. WT mice. (D) Data are representative of three independent experiments. Statistics: unpaired Student t test relative to WT (A) with a Welch correction in (C) and (D). *p < 0.05, ***p ≤ 0.001.
CD8 Tfc cells promote B cell Ab class switch
To determine the influence of IL-2–KO CD8 Tfc cells on B cell activities, we examined CD8 Tfc cell production of the cytokine IL-21, an effector cytokine produced by CD4 Tfh cells (10). Stimulation of CD8 Tfc cells yielded similar levels of IL-21 (average mean fluorescence intensity [MFI] of 120), as compared with IL-2–KO CD4 Tfh cells (average MFI of 136), and significantly more than naive WT cells or IL-2–KO non-Tfc cells (Fig. 6A). Consistent with higher IL-21 production, both CD4 Tfh cells and CD8 Tfc cells expressed higher levels of IL-21 mRNA as compared with their CXCR5−PD-1lo counterparts (Fig. 6B). We next evaluated whether CD8 Tfc cells secreted proteins capable of influencing B cell Ab class switching using in vitro culture assays. WT B cells were cultured with and without αIgM and αCD40 plus supernatant from activated T cells (Fig. 6C). Supernatant from IL-2–KO CD8 Tfc cells induced significant amounts of total IgG and IgG1 production by B cells, comparable to levels produced by B cells with immunized WT CD4 Tfh cell and IL-2–KO CD4 Tfh cell supernatants (Fig. 6D). These data confirm that IL-2–KO CD8 Tfc cells are capable of inducing Ab class-switch recombination independent of CD4 Tfh cells.
IL-2–KO CD8 Tfc cells promote Ab class switch by B cells. (A) IL-2–KO and WT lymphocytes were stimulated with PMA and ionomycin. CD8 and CD4 T cells were gated on the indicated populations and analyzed for IL-21 expression by flow cytometry. (B) IL-21 mRNA expression in sorted IL-2–KO CD4 Tfh or CD8 Tfc cells relative to IL-2–KO CD4 non-Tfh or CD8 non-Tfc cells, respectively. (C) Schematic describing the assay for T cell stimulation and B cell Ab induction. B cell supernatant was analyzed for total IgG and IgG1 by ELISA. (D) Total IgG concentration or IgG1 levels of stimulated B cells with and without stimulated IL-2–KO CD4 Tfh cells, IL-2–KO CD8 Tfc cells, or KLH-immunized WT (Imm. WT) CD4 Tfh cell supernatants were determined by ELISA. (E) Schematic describing IL-2–KO T cell transfer and B cell induction. Sorted IL-2–KO CD4 or CD8 T cells were adoptively transferred to TCRα-KO mice, immunized with KLH in CFA, and reimmunized with NP-KLH in IFA. (F) B220+GL-7+Fas+ GC B cells frequency and (G) B220intCD138+ plasma cell frequency from TCRα-KO recipient spleens. (H) IgG1, IgG2b, and IgG2a levels determined by ELISA from TCRα-KO recipient serum. (A, F, and G) Each symbol indicates an individual animal. Data are representative of four to six independent experiments. Statistics: unpaired Student t test relative to WT with a Welsh correction (A) and ordinary one-way ANOVA with select comparisons and a Bonferroni correction (D and F–H). ***p ≤ 0.001.
To next evaluate the impact of CD8 T cells on B cell responses in vivo, we adoptively transferred IL-2–KO CD4 T cells, CD8 T cells, or a combination of both into TCRα-KO mice that were then immunized with KLH and reimmunized with NP-KLH (Fig. 6E). Fourteen days posttransfer, Fas+GL-7+ GC B cell expansion was measured relative to PBS-treated TCRα-KO mice. Transferred IL-2–KO CD4 T cells alone produced significant GC B cell expansion, whereas transfer of IL-2–KO CD8 T cells alone did not. A combination of IL-2–KO CD4 and CD8 T cells yielded similar GC B cell frequency as compared with IL-2–KO CD4 T cells alone (Fig. 6F). IL-2–KO CD4 and CD8 T cells, when transferred independently, induced a similar plasma cell expansion. Transfer of IL-2–KO CD4 and CD8 T cells together induced significantly more plasma cells than either population alone (Fig. 6G). Interestingly, when transferred alone, CD8 T cells did not induce Ab class switching. However, when transferred with CD4 T cells, CD8 T cells promoted an increase in IgG1 and significantly increased IgG2b as compared with the transfer of CD4 T cells alone (Fig. 6H). Together, these data demonstrate that CD8 T cells act synergistically with CD4 T cells to enhance B cells differentiation and specific class switching. CD8 Tfc cells, therefore, have the potential to provide a helper-like interaction within the GC to facilitate B cell Ab production during systemic autoimmune disease.
Discussion
In this study, to our knowledge, we provide the first evidence that a new class of CD8 Tfc cells develop during autoimmune disease in the absence of functional Tregs. In the setting of inflammation and autoimmunity, CD8 Tfc cells acquire CD4 Tfh cell–like functionality by producing Tfh cell effector cytokines and coreceptors and promoting B cell Ab class switching. CD8 Tfc cells promote Ab class-switch recombination at a level comparable to CD4 Tfh cells in vitro and synergize with CD4 Tfh cell responses in vivo. Our findings concur with recent reports describing the generation of similar CD8 Tfc cells during chronic infection (20, 21, 23, 53).
CD8 Tfc cells affected class-switch recombination in B cells through a cell-secreted factor in the supernatant. One likely factor is the cytokine IL-21, as it is produced by IL-2–KO CD8 Tfc cells. IL-21 is known to induce plasma cell differentiation, Ig production, and class switching. In vitro–derived CXCR5+CD8 cells also produce IL-21 that promotes influenza-specific IgG production that is reduced in IL-21R–deficient B cells (22). In addition to the role of IL-21 in developing CD4 Tfh cell populations, other CD4 Tfh cell–secreted cytokines promote specialized Ab class switch, for example, IFN-γ supports IgG2a, and IL-4 supports IgG1 switching (54, 55). Although IL-2–KO disease has been described as a Th1-mediated disease (5), IFN-γ–mediated class switching to IgG2a was only detected when IL-2–KO CD4 T cells were transferred alone and was only moderately affected by cotransfer with CD8 T cells (Fig. 6H). In contrast, both CD8 depletion and cotransfer of CD4 and CD8 T cells most significantly impacted class switching to IgG1. Autoimmune interactions in the IL-2–KO mouse, and specifically in the GC, may be governed by a combination of cytokines, including IL-21 and IL-4, in contrast to the IFN-γ production found systemically. Additionally, elevated CD40L and ICOS expression by IL-2–KO CD8 Tfc cells was observed and may promote B cell function via direct cell/cell interactions. In IL-2–KO mice, CD8 T cells localize to the B cell follicle and can be identified within the GC. CD8 T cells migrate into proximity with B cells, providing the localization necessary for influencing GC B cell reactions.
We report that autoimmune disease is delayed in IL-2–KO mice in the absence of CD8 T cells. Expansion of CD8 Tfc cells is also delayed in comparison with the observed expansion of CD4 Tfh cells during disease progression. The kinetics suggest that CD4 T cell dysfunction proceeding CD8 Tfc cell expansion may promote a transition to more rapid, lethal autoimmunity. Our reported differences in the frequency of CD4 Tfh and CD8 Tfc cells in the IL-2–KO and scurfy mouse models also suggest a role for IL-2 in CD8 Tfc cell development. The absence of IL-2 contributes to a lymphoproliferative disorder in IL-2–KO mice that is not seen in scurfy mice, which may partially account for observed differences (48). However, IL-2 is known to suppress CD4 Tfh cell differentiation via Bcl6 expression in vivo (56). Thus, the difference in CD8 Tfc cell frequency between IL-2–KO mice and scurfy mice may be explained in part by the reduced numbers of IL-2–expressing cells in addition to differences in lymphoproliferation. As scurfy mice produce fairly normal levels of IL-2 (Fig. 4A, 47), other factors in addition to IL-2 loss likely contribute to CD8 Tfc cell development.
As a reduction or impairment of Tregs is the driving defect underlying autoimmunity in both IL-2–KO (2) and scurfy mice (47), the expansion of CD8 Tfc cells in both models is likely due, in part, to the breakdown in immune tolerance mechanisms that precedes autoimmune disease. During chronic inflammation and situations of high localized Ag, especially when immune regulation is compromised, CD4 Tfh cells expand (13, 16). Cellular expansion and inflammation occurs in the IL-2–KO mouse because of reduced function and frequency of Foxp3+ Tregs (2). Both CD4 and CD8 Tregs have been identified as essential regulators of GC tolerance (7, 8). The absence of CD4 Tfc regulatory cells has been shown to promote autoimmune disease and aging via increased CD4 Tfh cells, GC B cells, and Ab class switching (7, 57). Impaired Treg function may similarly allow for the development of CD8 Tfc cells during chronic inflammatory conditions.
Together, this study adds to a growing body of research supporting a helper role for CD8 T cells during chronic Ag exposure and inflammation, including chronic viral infections, cancer, and autoimmune disease. Our data provide a unique perspective on the role of CD8 T cells in GC interactions that may promote, amplify, or shift the autoimmune disease process. Future studies are needed to reveal the overlapping and distinct immune stages, roles, and influences of CD4 Tfh and CD8 Tfc cells during autoimmune disease progression. The identification of CD8 Tfc cell interactions within the GC provides many avenues for continuing research, including defining the contribution of CD8 Tfc cells to immunity and disease. An understanding of the types of Abs generated and the contribution to affinity maturation throughout the kinetics of CD8 Tfc cell development may unveil a new paradigm for GC reactions and a deeper insight into strategies for manipulating these processes.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank Dr. Jennifer Manilay for scientific advice and critical evaluation of the manuscript, Roy Hoglund and the staff members of the UC Merced Department of Animal Research Services for animal husbandry care, the UC Merced Stem Cell Instrumentation Foundry for assistance in cell sorting, and Anh Diep, Haword Cha, and Karina Arroyo for experimental assistance and technical support.
Footnotes
This work was supported by the National Institutes of Health Grant R00HL090706 to K.K.H., the National Heart, Lung, and Blood Institute Mentored Career Development Award K01HL130753 to A.E.B., an Aplastic Anemia and MDS International Foundation grant to K.M.V., and by the National Science Foundation Grant ACI-1429783 to T.J.L.
The sequences presented in this article have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE112540.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- GC
- germinal center
- HET
- heterozygous
- KLH
- keyhole limpet hemocyanin
- KO
- knockout
- LN
- lymph node
- MFI
- mean fluorescence intensity
- NP
- nitrophenylacetyl
- Tfc
- T follicular
- Tfh
- T follicular helper
- Treg
- T regulatory cell
- UC
- University of California
- WT
- wild-type.
- Received July 26, 2017.
- Accepted April 22, 2018.
- Copyright © 2018 by The American Association of Immunologists, Inc.
References
In this issue
