Dual TCRα expression poses an autoimmune hazard by limiting T_reg cell generation

Abstract

Despite accounting for 10–30% of the T cell population in mice and humans, the role of dual TCR-expressing T cells in immunity remains poorly understood. It has been hypothesized that dual TCR T cells pose an “autoimmune hazard” by allowing self-reactive TCRs to escape thymic selection. We revisited this hypothesis using the NOD murine model of type 1 diabetes (T1D). We bred NOD mice hemizygous at both TCRα and β (TCRα^+/− β^+/−) loci, rendering them incapable of producing dual TCR T cells. We found that the lack of dual TCRα expression skewed the insulin-specific thymocyte population toward greater regulatory T (T_reg) cell commitment, resulting in a more tolerogenic T_reg to conventional T (T_conv) cell ratio and protection from diabetes. These data support a novel hypothesis by which dual TCR expression can promote autoimmunity by limiting agonist selection of self-reactive thymocytes into the T_reg cell lineage.

Introduction

Type 1 diabetes (T1D) is an autoimmune disease characterized by immune-mediated destruction of insulin-producing pancreatic beta cells. T1D, like all autoimmune diseases, occurs due to failure of immune tolerance mechanisms to prevent or control self-reactive immune responses. In humans and NOD mice, disease is driven by intolerant insulin-specific CD4⁺ T cells (1–5). How insulin-specific CD4⁺ T cells escape tolerance is incompletely understood. We investigated dual TCR expression as a potential mechanism by which pathogenic CD4⁺ T cells escape tolerance.

Allelic exclusion ensures that most T cells only express a single recombined αβ TCR specificity. However, allelic exclusion is imperfect, and an estimated 10–30% of all T cells express two functionally recombined TCRα chains in mice and humans (6–9). TCRβ allelic exclusion is more stringent, with 1–3% expressing two TCRβ chains (6). Since the discovery of dual TCR T cells, there has been concern that they might promote autoimmunity (9). This typically relates to their potential to impair negative selection (8, 10–13), although dual TCR expression can also promote positive selection of otherwise unselected specificities (14–17). Many transgenic TCR mouse models support these mechanisms (10, 11, 13–17). However, most TCR transgenic models have unusually high fractions of dual TCR T cells, and earlier-than-normal expression of transgenic TCRs alters T cell development. The impact of dual TCR expression on thymic selection under normal, non-TCR transgenic conditions is unknown. Here, we explored the contribution of dual TCR expression to spontaneous autoimmune diabetes in NOD mice with a normal polyclonal TCR repertoire, contrasting WT NOD mice with NOD mice unable to make dual TCR T cells. To our surprise, we discovered a previously-unrecognized impact of dual TCR expression on agonist selection.

Materials and Methods

Mice

TCRα knockout (KO) NOD (NOD.129P2(C)-Tcra^tm1Mjo/DoiJ) mice were from Christophe Benoist and Diane Mathis via The Jackson Laboratory (strain 004444) (18). We generated TCRβ KO NOD mice using speed congenic technology to backcross the Tcrb^tm1Mom allele from C57BL/6 TCRβ KO mice (B6.129P2-Tcrb^tm1Mom/J) onto the NOD background (IDEXX) (19, 20). The presence of known insulin dependent diabetes (Idd) loci was verified by PCR (Supplemental Fig. 1A–B) and the absence of dual TCR T cells was confirmed by flow cytometry (Supplemental Fig. 1C–D). Mice were bred and housed in specific pathogen-free colonies at the University of Minnesota. All animal use was performed per the University of Minnesota Institutional Animal Care and Use Committee-approved protocols (1206A15325 and 1503-32409A).

Diabetes monitoring

Female NOD mice were monitored for diabetes weekly after 10 weeks of age. Mice were deemed diabetic following two consecutive blood-glucose readings >250 mg/dL (Diastix, Bayer Healthcare).

Islet Infiltration Analysis

Pancreata were harvested from 10-week-old pre-diabetic single TCR T cell and WT NOD female mice and prepared as described (21). Insulitis was determined manually using insulitis scoring scale: 0 = no insulitis, 1 = peri-insulitis, 2 = mild insulitis, 3 = moderate insulitis, 4 = severe insulitis. Islet-infiltrating CD3⁺, CD4⁺_, and Foxp3⁺ T cell enumeration was determined using Imaris 8 software (Bitplane) where T_reg cells were surface positive for CD4 or CD3 and nucleus positive for Foxp3.

Antibody Treatment

For PD-1 blockade experiments, 10-week-old single TCR T cell and WT NOD female mice were treated intraperitoneally (IP) with 250 μg anti–PD-1 (clone J43, Bio X Cell, West Lebanon, NH) on day 0 and with 200 μg on days 2, 4, 6, 8, and 10 (22). For transient depletion of CD25 positive cells, a single dose of 500 μg anti-CD25 (clone PC-61.5.3, Bio X Cell) was administered intraperitoneally at between 3.5–4 weeks of age (23).

Tetramers

All MHCII:peptide tetramers used in these experiments were generated at our institution as previously described (21). P8G and P8E tetramers were originally designed to have either glycine-glutamic acid (SHLVEALYLVCGGEG) or glutamic acid-glutamic acid (SHLVEALYLVCGEEG) at positions B:21–22 of the native InsB_9–23 sequence (SHLVEALYLVCGERG) (24).

Flow Cytometry

Thymus, PLN, and pancreata were harvested from pre-diabetic 10-week-old mice. Pancreata were dissociated using the Miltenyi Biotec Tumor Dissociation Kit (product 130-096-730) and the Gentle MACs dissociator followed by debris separation with Debris Removal Solution (product 130-109-398, Miltenyi Biotec). Cell characterization was determined by surface and intracellular cell marker expression by flow cytometry. Cell viability was determined with Live/Dead-Aqua viability dye (Biolegend). Analysis was performed on a BD LSR Fortessa flow cytometer (BD Biosciences). Data were analyzed with FlowJo Software version 8 (FloJo LLC).

Statistical Analysis

Statistical significance was determined using unpaired Student t-test or Mann-Whitney test (pancreas flow cytometry data). Differences in diabetes incidence were determined using log-rank (Mantel-Cox) tests. Data were analyzed using Prism 5 (GraphPad Software).

Results/Discussion

Hemizygosity at the TCRα locus protects NOD mice from diabetes

We generated NOD mice with targeted deletions at TCRα, TCRβ, or both loci. We then bred female NOD mice to be hemizygous at TCRα (TCRα^+/−), TCRβ (TCRβ^+/−), or both loci (TCRα^+/− β^+/−, hereafter “single TCR T cell mice”). We evaluated the incidence of spontaneous diabetes in each group, using WT NOD mice as controls (Fig. 1A). Single TCR T cell and TCRα^+/− NOD mice both displayed diabetes resistance, whereas TCRβ^+/− and WT NOD mice developed diabetes similarly (Fig. 1A). These data indicate that resistance to diabetes is associated with hemizygosity at the TCRα, but not the TCRβ locus. Therefore, effects of TCRβ hemizygosity that we have previously demonstrated, cannot be implicated (19). Two prior investigations of diabetes in TCRα^+/− NOD mice by Elliott and Altmann initially reported protection from diabetes, but their follow-up study found no difference between TCRα^+/− and WT NOD mice (25, 26). Interpretation of these studies is complicated by incomplete backcrossing and cyclophosphamide induction of disease in the first study as well as non-specific pathogen free (SPF) conditions and very low spontaneous diabetes incidence in the second; these concerns do not apply to our study (see Methods and Fig. S1A). We next evaluated pancreatic islet infiltration in 10-week-old female WT and single TCR T cell NOD mice. Islet infiltration was present in single TCR T cell NOD mice, though much less severe than in WT NOD mice (Fig. 1B). Together these data demonstrate that dual TCRα expression promotes increased islet infiltration and diabetes.

Fig. 1. Lack of dual TCRα expression imparts resistance to diabetes in NOD mice.

A) Female single TCR T cell (TCRα^+/− β^+/−), TCR α^+/−, TCRβ^+/−, and WT NOD mice were monitored for diabetes. Data were accumulated over the course of two years from multiple litters. B) Insulitis score was manually determined using immunofluorescent microscopy. C) Diabetes incidence following transient ablation of CD25-expressing cells. For diabetes incidence, p values were calculated using Log-rank (Mantel-Cox) tests and are based on comparisons with WT NOD controls.

Long term resistance to diabetes in single TCR T cell mice is not mediated by PD-1, by anergy, or antigen sequestration

Long-term tolerance to diabetes has been demonstrated to depend on the programmed death-1 (PD-1) signaling pathway (22, 27, 28). To determine whether diabetes resistance among single TCR T cell NOD mice depends on this pathway, we administered anti-PD-1 blocking antibody to 10-week-old female WT or single TCR T cell NOD mice. Blocking PD-1 induced diabetes in 5 of 7 WT NOD mice, but only 3 of 10 single TCR T cell NOD mice (Supplemental Fig. 2A). We also observed no difference in PD-1 expression in bulk CD4⁺ or insulin-specific T cells derived from the pancreas-draining lymph node (PLN) (Supplemental Fig. 2B). Further characterization of the bulk and insulin-specific PLN CD4⁺ T cells revealed equivalent T cell antigen experience (as measured by CD44 expression) and equivalent FR4 and CD73 expression (markers of anergy) (Supplemental Fig. 2C–D) (29). These data demonstrate that insulin-specific CD4⁺ T cells encounter antigen and are chronically stimulated equivalently in both groups of mice. Taken together, these findings suggest that diabetes resistance in single TCR T cell NOD mice does not depend on the PD pathway and is not associated with increased anergy or antigen sequestration. We therefore explored other potential mechanisms of tolerance.

Depletion of regulatory T cells abrogates diabetes protection in single TCR T cell NOD mice

Regulatory T (T_reg) cells are essential mediators of peripheral immune tolerance and protection from T1D in humans and mice (30–35). We asked whether T_reg cells are responsible for diabetes protection in single TCR T cell mice using early ablation of CD25-expressing cells (32). Anti-CD25 treatment induced diabetes in all the WT NOD mice by 20 weeks of age. Importantly, 5 of 7 single TCR T cell NOD mice also developed diabetes by 20 weeks of age; in sharp contrast to untreated (1/10) and anti-PD-1 treated (3/10) single TCR T cell NOD mice (Fig. 1A, 1C, and Supplemental Fig. 2A). These findings demonstrate a) that single TCR T cell NOD mice can develop diabetes and b) that T_reg cells help to maintain tolerance. We then focused on how dual TCR expression affects antigen-specific T_reg cell populations.

Single TCR T cell NOD mice have an increased insulin-specific T_reg:T_conv ratio

We characterized T cell lineage commitment in the PLNs of 10-week-old single TCR T cell and WT NOD mice by flow cytometry. Both groups had similar numbers of bulk, insulin-specific, and foreign-specific (HEL_11–25) CD4⁺ T cells, indicating resistance to diabetes is not a result of a smaller insulin-specific T cell population (Fig. 2A). In contrast, the insulin-specific T_reg population was 3-fold larger in single TCR T cell mice relative to WT NOD mice, while bulk and HEL_11–25-specific T_reg cell numbers were similar (Fig. 2B). When considered together these data reveal a 3-fold increased insulin-specific T_reg to conventional CD4⁺ T cell (T_reg:T_conv) ratio. Diabetes is largely driven by insulin-specific conventional T (T_conv) cell responses (1–5), and many studies have shown that expansion of the insulin-specific T_reg population can prevent diabetes (36–38). These studies indicate that the balance between T_reg:T_conv insulin-specific cells is crucial to disease. Altogether, these data favor insulin-specific T_reg cells as a primary mediator of diabetes resistance in single TCR T cell NOD mice.

Fig. 2. Lack of dual TCR expression results in higher insulin-specific T_reg:T_eff ratios.

PLN were collected from 10-week-old WT or single TCR T cell NOD female mice and analyzed by flow cytometry. A) Total CD4⁺ T cells, InsB_9–23-tetramer specific, or HEL_11–25-tetramer-specific T cells. B) Total number of bulk CD4⁺, InsB_9–23-tetramer-specific, or HEL_11–25-tetramer specific T_reg cells. Each data point represents the average number of cells per mouse from the combined PLN of 5 mice. p values were determined using unpaired Student’s t-test.

We then enumerated T_reg cells in the pancreas by immunohistochemistry. Single TCR T cell NOD mice had significantly fewer CD3⁺ T cells present in or around islets and a significantly higher T_reg:CD3⁺ T cell ratio per islet relative to WT NOD mice (Fig. 3A–B). We further verified the presence of T_reg cells in and around islets via immunostaining of tissue sections with CD4 and FOXP3 antibodies (Fig. 3C). Collectively, the data presented in Figures 2 and 3 support a model in which skewing of insulin-specific T cells to the T_reg lineage promotes diabetes resistance in single TCR T cell NOD mice.

Fig. 3. Lack of dual TCR expression results in larger T_reg:T_conv ratios in the pancreas.

A) Total number of CD3⁺ T cells per islet in WT or single TCR T cell NOD mice. B) Bulk T_reg:CD3⁺ cell ratios of cryopreserved pancreas slices were determined by immunofluorescent microscopy. C) Representative images demonstrating T_reg islet infiltration from WT and Single TCR T cell NOD mice. p values were determined using unpaired Student’s t-test.

Lack of dual TCRα expression increases agonist selection of self-reactive T cells

We postulated that the T_reg lineage skewing in the single TCR T cell mice originates in the thymus. We therefore contrasted thymocyte development stages of the single TCR T cell, TCRα^+/−, and WT NOD mice. Inclusion of TCRα^+/− NOD mice in these experiments allowed us to separate effects on T cell development resulting from hemizygosity at TCRβ locus, and therefore not involved in diabetes pathogenesis, from those of hemizygosity at the TCRα locus. We found that single TCR T cell and TCRα^+/− NOD mice had a decreased proportion of single positive (SP) thymocytes relative to WT NOD mice (Fig. 4A). We separated the SP population into CD4⁺ (non-T_reg), CD8⁺, and T_reg cells and found that both single TCR T cell and TCRα^+/− NOD mice had lower frequencies of CD4⁺ and CD8⁺ cells than WT NOD mice, but an equivalent proportion of T_reg cells. This resulted in a higher T_reg:SP ratio, similar to the pancreas and insulin-specific T cell population in the PLN, suggesting that this ratio is established early in the thymus (Fig. 4B and C). We scrutinized positive selection using TCRβ and CD5 expression as surrogate markers for TCR signaling and progression through positive selection (where TCRβ^lo CD5^lo -pre-selection, TCRβ^lo CD5^int-initiating positive selection, TCRβ^int CD5^hi-undergoing positive selection, and TCRβ^hi CD5^hi-post-selection) (Fig. 4D) (39). We found that single TCR T cell and TCRα^+/− double positive (DP) thymocytes were more likely to be classified as pre-selection and less likely to initiate and undergo positive selection relative to WT DP thymocytes, indicating less efficient positive selection (Fig. 4D). Similarly, the proportion of post-selection DP thymocytes was lower in TCRα^+/− NOD mice, and all but one single TCR T cell NOD mouse trended lower than WT (Fig. 4D). The reduction in total SP thymocytes could be attributed to decreased efficiency of positive selection during the DP stage due to increased failure to recombine a functional TCR, consistent with prior work from our group and others (14, 19). However, since T_reg committed thymocytes are derived from the CD4 SP thymocyte subset, this population would be similarly affected. We suggest that this effect in mice lacking dual TCRα expression is counteracted by the increased efficiency of agonist selection into the T_reg lineage among medullary thymocytes.

Fig. 4. Lack of dual TCR expression promotes thymic agonist selection.

Thymi were harvested from 10-week-old WT, single TCR T cell, or TCRα^+/− NOD female mice. Thymocyte development and selection were analyzed by flow cytometry. A) Thymocyte development efficiency was determined by contrasting proportions of thymocytes at each development stage (DN, DP and SP). B) Breakdown of the SP thymocyte population into CD4⁺, CD8⁺, and T_reg subsets. C) T_reg:SP ratios. D) Positive selection efficiency for each group was determined using TCRβ and CD5 surface expression. Sample gating strategy is depicted on the right. E) The proportion of InsB_9–23-tetramer specific thymocytes in the CD4⁺ SP subset. F) Mean fluorescent intensity (MFI) of CD5 in CD4⁺, T_reg, and InsB_9–23-tetramer-specific cells in the SP subset. CD5 MFI is represented as fold increase over CD4⁺ SP thymocytes. G) Activated caspase 3 was measured in the signaled (CD5⁺ TCRβ⁺) medullary (CCR7⁺) CD4⁺ subset in each experimental group by flow cytometry. A–F) data are from two independent experiments. p values were determined using unpaired Student’s t test. *, **, and *** indicate p≤0.05, p≤0.01 and p≤0.001 respectively relative to the CD5 MFI in the corresponding CD4⁺ thymocyte population.

Focusing on antigen-specific cells, we found that insulin-specific CD4⁺ thymocytes accounted for a similar proportion of the repertoire in the three groups of mice (Fig. 4E). We measured the mean fluorescent intensity (MFI) of CD5 (a reliable surrogate for TCR signal strength) (40), on CD4⁺ SP, T_reg, and insulin-specific thymocytes from all groups. Insulin-specific thymocytes, much like T_reg thymocytes, expressed higher levels of CD5 than did CD4⁺ SP thymocytes, indicating that they have received strong TCR signals during thymic selection (Fig. 4F). Thymocytes that receive strong TCR signals are either deleted by apoptosis or undergo agonist selection to become T_reg cells, invariant natural killer T cells, or intraepithelial lymphocytes. We therefore sought clues to determine whether these CD4 SP thymocytes were undergoing apoptosis or agonist selection. Caspase 3 is a member of the cysteine-aspartic acid family of proteases that is activated during apoptosis and therefore serves as a marker for apoptotic cell death. Importantly, agonist selection of strongly self-reactive thymocytes occurs in the medulla. As such, we measured activated caspase 3 in “signaled” (bulk CD4⁺ CD5⁺ TCRβ⁺) medullary (CCR7⁺) thymocytes by flow cytometry. We observed a lower proportion of caspase 3 activation in single TCR T cell and TCRα^+/− NOD mice relative to WT NOD mice in the medullary fraction (Fig. 4G). The higher T_reg:SP ratio present in⁻ NOD mice hemizygous for TCRα and our observations that CD5 expression is high among the insulin-specific thymocytes yet apoptosis is decreased among the “signaled” CD4 SP medullary thymocytes, support the hypothesis that the increase in insulin-specific T_reg cell commitment arises due to enhanced agonist selection into the T_reg lineage, and that this effect, rather than enhanced negative selection, underlies their diabetes resistance.

Our findings suggest that a subset of insulin-reactive CD4⁺ T cells express TCRs which, when expressed alone, favor T_reg cell commitment. The notion that certain TCRs preferentially direct T_reg cell commitment has previously been described, including TCRs with high affinity for InsB_9–23 (41–43). Increasing evidence suggests that thymically-derived T_reg cells are predominantly selected by Aire-expressing medullary thymic epithelial cells (mTECs) (41, 42). InsB is known to be both regulated by Aire and expressed by mTECs in the thymus (44). We hypothesize that when T_reg-biasing, insulin-specific TCRs are expressed in the context of a second TCR, the overall TCR avidity is reduced, favoring commitment to the T_conv lineage over the T_reg lineage. This would appear to be at odds with the findings of Tuovinen et al. which suggests the peripheral T_reg population is enriched for dual TCR T cells in humans (45). However, their observations are based on a single dual Vα population (Vα2⁺ Vα12⁺) and it is not known if they are thymically-derived or peripherally-induced T_reg cells. If other thymocyte populations reactive against Aire-regulated self-antigens behave similarly, one would expect a similar skewing toward a higher T_reg:CD4⁺ cell ratio among these populations. Future studies will need to be performed to investigate how dual TCR expression and thymic antigen expression patterns combine to influence T cell lineage commitment.

Altogether, our findings thus reveal a novel pathway of dual TCR T cell-mediated induction of autoimmune disease resulting from reduced agonist selection into the T_reg cell lineage, altering the composition of the self-reactive T cell population to a less tolerogenic, more autoimmune-prone phenotype. Furthermore, our findings support the idea that interventions designed to improve self-antigen-specific T_reg cell commitment and maintenance are a logical approach to prevent or treat T1D and other autoimmune diseases.