MHC class II (MHCII)–influenced CD4+ T cell differentiation and function play critical roles in regulating the development of autoimmunity. The lack of hematopoietic MHCII causes autoimmune disease that leads to severe wasting in syngeneic recipients. Using murine models of bone marrow transplantation (BMT), we find that MHCII−/−→wild-type BMT developed disease, with defective development of innate memory phenotype (IMP, CD44hi/CD62Llo) CD4+ T cells. Whereas conventional regulatory T cells are unable to suppress pathogenesis, IMP CD4+ T cells, which include conventional regulatory T cells, can suppress pathogenesis in MHCII−/−→wild-type chimeras. The functional development of IMP CD4+ T cells requires hematopoietic but not thymic MHCII. B cells and hematopoietic CD80/86 regulate the population size, whereas MHCII expression by dendritic cells is sufficient for IMP CD4+ T cell functional development and prevention of pathogenesis. Furthermore, the absence of Tec kinase IL-2–inducible T cell kinase in MHCII−/− donors leads to preferential development of IMP CD4+ T cells and partially prevents pathogenesis. We conclude that dendritic cells-MHCII and IL-2–inducible T cell kinase regulate the functional development of IMP CD4+ T cells, which suppresses the development of autoimmune disorder in syngeneic BMTs.

Bone marrow transplantation (BMT) is widely used therapeutically; however, graft-versus-host disease (GVHD) affects posttransplantion recovery, particularly in allogeneic BMTs (1). Syngeneic transplantation in mice has revealed that MHC class II (MHCII)–CD4 interactions play critical roles in the establishment of successful graft acceptance (2, 3). Transplantation of bone marrow lacking expression of MHCII into lethally irradiated syngeneic wild-type (WT) recipients (MHCII−/−→WT) evolves into systemic autoimmune-like GVHD, with lethal colitis and severe wasting, suggesting an impaired hematopoietic MHCII–mediated peripheral regulatory mechanism (3), which is to date unclear.

Others and we have previously described a subset of CD4+ T cells that have a memory-like phenotype and rapidly produce IFN-γ upon stimulation, referred to as innate memory phenotype (IMP; CD44hiCD62Llo in the periphery, and CD44hiCD122+ in the thymus) CD4+ T cells, exemplified in mice lacking the tyrosine kinase IL-2–inducible T cell kinase (Itk) (4, 5). The developmental requirement and physiological function of IMP CD4+ T cells are unknown. Memory-like cells have been better described in the CD8+ T cell compartment, with elevations observed in a variety of genetically manipulated mice such as those lacking Itk, CREB-binding protein, DNA-binding protein inhibitor (Id3), or Krüppel-like factor 2 (612), expressing an Slp-76 mutant that leads to impaired Itk downstream signaling activation (13), and those overexpressing T cell specific transcriptional factor 7, β-catenin (14), or promyelocytic leukemia zinc finger protein (15). We previously showed that hematopoietic MHC class I is required for the functional development of IMP CD8+ T cells (16). However, whether MHCII performs similar function for innate memory differentiation in CD4+ T cells remains to be investigated.

In this study, we examined the cellular and molecular mechanisms of IMP CD4+ T cell functional development. We used reciprocal bone marrow transplantation using MHCII−/− and WT mice as donor or recipient, generating mice with predominantly naive or IMP CD4+ T cells, which are transcriptomically similar to their counterparts in WT mice. These MHCII−/−→WT BMT chimeras lack IMP CD4+ T cells and suffer significant weight loss with reduced IL-10 and increased TNF-α production by T cells. The IMP CD4+ T cell populations include multiple T helper subsets and can prevent pathogenesis in MHCII−/−→WT chimeras. MHCII re-expression on dendritic cells (DCs) in MHCII−/− background completely rescues the development of IMP CD4+ T cells and suppresses the pathogenesis. Finally, we show that Itk has the potential to be a therapeutic target allowing the expansion of IMP CD4+ T cells in BMT lacking hematopoietic MHCII.

All mice were on a C57BL/6 background. MHCII−/− (B6.129S2-H2dlAb1-Ea/J), Thy1.1 (B6.PL-Thy1a/CyJ), and CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ), nude (B6.129(SJL)-Foxn1tm1.1Dmsu/J), Rag1−/− (B6.129S7-Rag1tm1Mom/J), μMT (B6.129S2-Ighmtm1Cgn/J), and CD80/86 double-knockout (B6.129S4-Cd80tm1ShrCd86tm1Shr/J) mice were from The Jackson Laboratory (Bar Harbor, ME). CD11c promoter-driven MHCII re-expressing Tg(CD11c-MHCII)/MHCII−/− (referred to as MHCIIDC) mice were previously described (17). Itk−/−MHCII−/− mice were generated by crossing Itk−/− and MHCII−/− mice. All experiments were approved by the Office of Research Protection’s Institutional Animal Care and Use Committee at The Pennsylvania State University and Cornell University.

Bone marrow chimeras were generated as previously described (4) (illustrated in Supplemental Fig. 1A). Briefly, 6- to 8-wk-old recipient mice were pretreated with acid water (pH 2–3) containing 1 mg/ml gentamicin sulfate solution (Sparhawk Laboratories, Lenexa, KS) 1 wk prior to lethal gamma irradiation (950 cGy), followed by retro-orbital injection with 107 donor bone marrow cells (2–4 mo old, same gender as recipients). Eight to 10 wk after bone marrow reconstitution, recipients were analyzed by gating on CD4+ T cells of donor origin (based on congenic marker CD45.1, CD45.2, or Thy1a) for IMP surface marker CD44/CD62L expression and ability of IFN-γ production (Supplemental Fig. 1B). Chimeric mice were weighed at indicated time points after transplantation at the same time each day.

All fluorochrome-conjugated Abs used are listed in a “fluorochrome-target” format as follows: eFluor 450-CD122, PE-Foxp3 allophycocyanin-CD4, PerCP-eFluor 710-TNF-α, PE-Cy7-Thy1.1, PE-Cy7-CD62L, and PE-Cy7-IFN-γ were from eBioscience (San Diego, CA); V500-CD44, FITC-CD45.1, FITC-TCRβ, PE-CD25, Alexa Fluor 700-CD45.2, Alexa Fluor 700-CD62L, PE-Cy5-CD44, PE-Cy7-CD4, and allophycocyanin-Cy7-TCRβ were from BD Biosciences (San Diego, CA); and PE-Texas Red-CD4 and CD8 were from Invitrogen (Carlsbad, CA). PE-PBS-57 (analog of α-galactosylceramide)–loaded CD1d tetramer was from the National Institute of Allergy and Infectious Diseases Tetramer Facility. Cells were stained for flow cytometric analysis as previously described (16). Briefly, live cells are incubated with Fc block (eBioscience) in 2% FBS containing PBS, followed by staining with indicated Abs against surface markers; to stain cytokines, cells were further fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), permeabilized, and stained with cytokine Abs using PBS containing 0.3% saponin (Sigma-Aldrich). Foxp3 staining was performed with Foxp3 staining buffer kit (eBioscience) following the manufacturer's instruction. Flow data were acquired a on an FC500 (Beckman Coulter, Brea, CA) or LSRII system (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR).

WT naive (CD44loCD62Lhi) and WT IMP (CD44hiCD62Llo) TCRβ+CD4+ T cells from WT mice, chimeric naive (CD45.2+CD44loCD62Lhi, CD45.2+ MHCII−/−→CD45.1+ WT chimeras sorted for donor naive cells), and chimeric IMP (CD45.2CD44hiCD62Llo, CD45.1+ WT→CD45.2+ MHCII−/− chimeras sorted for donor IMP cells) TCRβ+CD4+ T cells of donor source from bone marrow chimeras were sorted on an influx cell sorter (Cytopeia, Seattle, WA), and cells with purity >95% were used for all experiments. For regulatory cell transfer experiments, conventional regulatory T cells (TCRβ+CD4+CD25hi) and IMP CD4+ T cells (TCRβ+CD4+CD44hiCD62Llo) were sorted from WT mice (Thy1.1+) on a FACSAria Cell Sorter (BD Biosciences). Unless specified otherwise, 0.2–0.3 × 106 cells/injection were used.

Cells were flow sorted as described above. Total RNA was isolated from sorted WT naive, WT IMP, chimeric (MW: MHCII−/−→WT) naive, and chimeric (WM: WT→MHCII−/−) IMP CD4+ T cells using an RNeasy Plus Mini kit (Qiagen, Valencia, CA), amplified using a MessageAmp Premier RNA amplification kit (Life Technologies, Grand Island, NY), followed by examination on an Affymetrix mouse 430.2 array (Affymetrix, Santa Clara, CA). Microarray data were processed, analyzed, and rendered using GeneSpring version 12 (Agilent Technologies, Santa Clara, CA) as previously described (16). All values were further normalized to the average value of each gene in WT naive CD4+ T cells. Data have been deposited into the National Center for Biotechnology Information’s Gene Expression Omnibus repository (http://www.ncbi.nlm.nih.gov/gds) under accession number GSE46892.

To detect cytokine production using flow cytometry, splenocytes were left unstimulated or stimulated with 100 ng/ml PMA (Sigma-Aldrich), 0.5 μM ionomycin (Sigma-Aldrich), and 10 μg/ml brefeldin A (Sigma-Aldrich) for 4 h as previously described (4, 7). To examine T cell–derived cytokine secretion, total splenocytes were stimulated with 1 μg/ml anti-CD3ε and anti-CD28 Abs (eBioscience) for 3 d and supernatants were examined for cytokines using a Milliplex multiplex system (EMD Millipore, Billerica, MA) following the manufacturer’s instructions.

An unpaired two-tailed Student t test and two-way ANOVA were performed using GraphPad Prism (GraphPad Software, San Diego, CA). Group average was plotted with error bar indicating SEM. Differences with probability of p < 0.05 were considered statistically significant.

Naive or conventional αβ CD4+ T cells are characterized as CD44lo/CD62Lhi (18) and require the expression of MHCII on thymic epithelium for development (19, 20). The memory CD4+ T cell population (CD44hi and/or CD62Llo) is heterogeneous and is composed of both genuine Ag-specific memory T cells, as well as memory phenotype cells that arise independently of Ag stimulation (18, 21); in the absence of Itk, the latter selectively develops in naive mice (4). Hematopoietic MHC class I is required and sufficient for the development of innate memory CD8+ T cells (9, 16). To investigate this in the CD4+ T cell compartment, we generated bone marrow chimeras to allow T cell development in the absence of either hematopoietic or thymic epithelial MHCII. Compared to the WT bone marrow control, transfer of MHCII−/− bone marrow into WT mice (MHCII−/−→WT) gave rise to predominantly naive CD4+ T cells, with defects in the development of functional IMP CD4+ T cells (CD44hiCD62Llo IFN-γ producers). In contrast, transfer of WT bone marrow into MHCII−/− mice (WT→MHCII−/−) gave rise to predominantly IMP CD4+ T cells (Fig. 1A, 1B). Of note, the thymic IMP CD4+ T cells, characterized as CD44hiCD122+, selectively developed in the absence of thymic MHCII (WT→MHCII−/− chimeras, Supplemental Fig. 2). To compare naive and IMP CD4+ T cells derived via this process to those from WT mice, we sorted MHCII−/−→WT (MW) naive and WT→MHCII−/− (WM) IMP CD4+ T cells and compared their gene expression profiles to nonmanipulated WT counterparts. We found few genes differentially expressed in cells from the chimeric mice compared with WT counterparts (Fig 1C). Hierarchical clustering of genes with >2-fold change between at least two groups also classified chimeric naive and IMP CD4+ T cells as similar to their nongrafted naive and IMP counterparts, respectively (Fig. 1D). Thus, the bone marrow chimeras can be considered as models to generate separate populations of naive and IMP CD4+ T cells in vivo. The development of IMP CD4+ T cells, but significantly diminished naive CD4+ T cells in the absence of thymic MHCII in the WT→MHCII−/− chimeras, suggested that IMP CD4+ cells can bypass thymic selection. To confirm this, we used athymic nude mice in similar experiments. We found that nude recipients of WT bone marrow (WT→nude) gave rise to functional IMP CD4+ T cells, suggesting that these cells can develop in the absence of a thymus (Fig. 1E, 1F). We do note that although we did not observe this in WT→WT chimeras, it is possible that donor-derived IMP CD4+ T cells expand in the nude environment and repopulated these compartments.

FIGURE 1.

IMP CD4+ T cell development requires hematopoietic MHCII but not the thymus. Bone marrow chimeras were generated as indicated and donor CD4+ T cells were analyzed. (A) Flow cytometric analysis of expression of CD44, CD62L, and IFN-γ. Data represent results of more than five independent experiments. (B) Percentages and numbers of naive (upper) and IMP (lower, with percentage of IFN-γ–secreting proportion) CD4+ T cells in indicated chimeras. Data were combined from more than three independent experiments. The p values were determined by Student t test. (C) Cells from WT mice or of donor origin from the indicated chimeras were flow sorted for microarray analysis. Volcano plots of genes in CD4+ T cells from MHCII−/−→WT (MW) naive, WT→MHCII−/− (WM) IMP, and WT IMP CD4+ T cells are compared as shown. Quantile normalized gene expression (to average level of WT naive cells) were filtered for >2-fold change (corrected p < 0.05) between at least two groups. (D) Hierarchical clustering of genes expressed by naive and IMP CD4+ T cells from chimeric and WT mice. (E) Bone marrow chimeric mice were generated as indicated, and splenocytes of donor origin were analyzed for CD44, CD62L, and IFN-γ expression. (F) Percentages (left) and number (middle) of IMP CD4+ T cells and percentage of CD44hi IFN-γ–producing CD4+ T cells (right) in indicated chimeric mice. The p values were determined by Student t test.

FIGURE 1.

IMP CD4+ T cell development requires hematopoietic MHCII but not the thymus. Bone marrow chimeras were generated as indicated and donor CD4+ T cells were analyzed. (A) Flow cytometric analysis of expression of CD44, CD62L, and IFN-γ. Data represent results of more than five independent experiments. (B) Percentages and numbers of naive (upper) and IMP (lower, with percentage of IFN-γ–secreting proportion) CD4+ T cells in indicated chimeras. Data were combined from more than three independent experiments. The p values were determined by Student t test. (C) Cells from WT mice or of donor origin from the indicated chimeras were flow sorted for microarray analysis. Volcano plots of genes in CD4+ T cells from MHCII−/−→WT (MW) naive, WT→MHCII−/− (WM) IMP, and WT IMP CD4+ T cells are compared as shown. Quantile normalized gene expression (to average level of WT naive cells) were filtered for >2-fold change (corrected p < 0.05) between at least two groups. (D) Hierarchical clustering of genes expressed by naive and IMP CD4+ T cells from chimeric and WT mice. (E) Bone marrow chimeric mice were generated as indicated, and splenocytes of donor origin were analyzed for CD44, CD62L, and IFN-γ expression. (F) Percentages (left) and number (middle) of IMP CD4+ T cells and percentage of CD44hi IFN-γ–producing CD4+ T cells (right) in indicated chimeric mice. The p values were determined by Student t test.

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MHCII−/−→WT syngeneic chimeras develop systemic autoimmune-like GVHD, with lethal severe wasting (as shown in Fig. 2A), suggesting an impaired hematopoietic MHCII–mediated peripheral regulatory mechanism (3). In contrast, WT→MHCII−/− bone marrow chimeras with predominant IMP CD4+ T cells do not develop such wasting. We examined cytokines produced downstream of T cell activation processes that may be altered between the MHCII−/−→WT chimeras and the WT→MHCII−/− chimeras, compared with WT→WT controls, and that correlated with disease occurrence. Aside from a significant reduction in IL-10 and increase in TNF-α production in MHCII−/−→WT chimeras (Fig. 2B), we did not find any patterns among Th1/Th2/Th17-type cytokines examined (Supplemental Fig. 3A). It is likely that other cells also contributed to this pattern following the initial T cell activation response. However, analysis of CD4+ T cells originated from MHCII−/−→WT donors revealed elevated TNF-α production and defective IL-10 secretion under TcR activating conditions, whereas donor-derived CD4+ T cells of WT→MHCII−/− chimeras appeared similar to those in WT→WT controls (Supplemental Fig. 3B). When we analyzed the transcriptomes of IMP CD4+ T cells, we found that they exhibit elevated expression of multiple master transcription factors for T helper sublineages, including AhR/c-MAF (driving IL-10 expression in Tr1), T-bet (Tbx21, Th1), Foxp3/Helios (Foxp3/Ikzf2, natural regulatory T cells), and PU.1 (Sfpi-1, Th9), and a slight increase in GATA3 (Th2) and retinoic acid–related orphan receptor γt (Rorc, Th17) (Fig. 2C). Correspondingly, IMP CD4+ T cells carry a mosaic T helper cytokine profile at the transcriptional level, with significant expression of both IL-4 (Th2) and IFN-γ (Th1), slightly enhanced IL-17A/IL-21 (Th17), and significantly increased IL-10 (Tr1), albeit little TGF-β (Fig. 2D). Thus, the IMP population of CD4+ T cells likely includes memory and memory-like cells for a number of different lineages, but prominent among them are cells that have a regulatory phenotype, suggesting a mechanism for their effect in vivo.

FIGURE 2.

Systemic defect of Treg function in mice lacking IMP CD4+ T cells. (A) Body weight of indicated bone marrow chimeras; n = 5. The p values were determined by a two-way ANOVA. (B) IL-10 and TNF-α secreted by anti-CD3/CD28–stimulated splenocytes from the indicated chimeric mice. Data were combined from more than three independent experiments. The p values were determined by Student t test. (C) Expression of master transcription factors in chimeric and WT naive or IMP CD4+ T cells. (D) Expression of cytokines in chimeric and WT naive or IMP CD4+ T cells. (E) Cells of donor origin in indicated chimeras were analyzed. Representative plots of Foxp3 expression by donor CD4+ T cells in spleens of indicated chimeric mice (upper) and summary of percentages (lower left) and numbers of (lower right) conventional Foxp3+ Tregs. Data were combined from more than three independent experiments. The p values were determined by Student t test. (F) Bone marrow chimeras were generated as described, and 0.2–0.3 × 106 conventional Tregs (+ Treg) or IMP CD4+ T cells (+ IMP CD4) were retro-orbitally injected 1 wk after bone marrow transplantation; n = 5. The p values were determined by a two-way ANOVA. *p < 0.05 by Student t test, comparing “+ IMP CD4” to “WT→WT” group of matched time point. (G) IMP CD4+ T cells (0.2–0.3 × 106) were given weekly to MHCII−/−→WT chimeras until sacrifice; n = 5. The p values were determined by a two-way ANOVA.

FIGURE 2.

Systemic defect of Treg function in mice lacking IMP CD4+ T cells. (A) Body weight of indicated bone marrow chimeras; n = 5. The p values were determined by a two-way ANOVA. (B) IL-10 and TNF-α secreted by anti-CD3/CD28–stimulated splenocytes from the indicated chimeric mice. Data were combined from more than three independent experiments. The p values were determined by Student t test. (C) Expression of master transcription factors in chimeric and WT naive or IMP CD4+ T cells. (D) Expression of cytokines in chimeric and WT naive or IMP CD4+ T cells. (E) Cells of donor origin in indicated chimeras were analyzed. Representative plots of Foxp3 expression by donor CD4+ T cells in spleens of indicated chimeric mice (upper) and summary of percentages (lower left) and numbers of (lower right) conventional Foxp3+ Tregs. Data were combined from more than three independent experiments. The p values were determined by Student t test. (F) Bone marrow chimeras were generated as described, and 0.2–0.3 × 106 conventional Tregs (+ Treg) or IMP CD4+ T cells (+ IMP CD4) were retro-orbitally injected 1 wk after bone marrow transplantation; n = 5. The p values were determined by a two-way ANOVA. *p < 0.05 by Student t test, comparing “+ IMP CD4” to “WT→WT” group of matched time point. (G) IMP CD4+ T cells (0.2–0.3 × 106) were given weekly to MHCII−/−→WT chimeras until sacrifice; n = 5. The p values were determined by a two-way ANOVA.

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Conventional Foxp3-expressing regulatory CD4+ T cells (Tregs) are well-known suppressors of multiple autoimmune diseases in both humans and mice (2224). We found that MHCII−/−→WT chimeric mice had significant reductions in Foxp3+ Tregs as a percentage of total CD4+ T cells, and this percentage was significantly higher in WT→MHCII−/− chimeras (Fig. 2E), confirming the presence of a Treg transcriptome signature in the IMP CD4+ T cells. Surprisingly, however, despite the higher percentage of Foxp3+CD4+ T cells, the number of Tregs in the WT→MHCII−/− chimeras was the same as that seen in MHCII−/−→WT chimeras that developed disease (Fig. 2E). These data suggest that in the MHCII−/−→WT chimeras, there is a lack of a regulatory compartment that results in disease, although the type of immunosuppressive T cell defect in these mice is unclear. It is possible that peripheral MHCII activates Tregs in WT→MHCII−/− chimeras, and thus these low numbers of Tregs can prevent pathogenesis but not those in MHCII−/−→WT chimeras; however, transferring Rag−/− splenocytes as APCs into MHCII−/−→WT chimeras to compensate for the lack of MHCII-expressing APCs in the periphery had a very subtle effect in regulating the disease development (Supplemental Fig. 4A).

To further investigate whether conventional Tregs (Foxp3-expressing) or IMP CD4+ T cells were responsible for the lack of suppression and subsequent autoimmune disorder in the MHCII−/−→WT chimeras, we sort-purified conventional Tregs (CD4+TCRβ+CD25hi) (most CD25hiCD4+ T cells are Foxp3+ (25), and there is >95% of CD25hiCD4+ T cells expressing high levels of Foxp3 in our WT mouse colony; Supplemental Fig. 4B) and IMP CD4+ T cells (CD4+TCRβ+CD44hiCD62Llo) from WT mice and transferred them into MHCII−/−→WT chimeras to determine whether they could prevent wasting. Transfer of IMP CD4+ T cells 1 wk after BMT significantly attenuated weight loss of MHCII−/−→WT chimeras; in contrast, transfer of equal numbers of Tregs (from the same donors) did not (Fig 2F). However, a single transfer of IMP CD4+ T cells at the early stage did not completely abrogate disease, as MHCII−/−→WT chimeric recipients eventually started losing significant amounts of weight by 8 wk. Our previous work indicated that IMP CD4+ T cells have a half-life of ∼14 d in vivo (4), and so it was possible that the incomplete rescue was due to turnover of the transferred IMP CD4+ T cells. We therefore transferred IMP CD4+ T cells once a week consecutively for 8 wk to ensure the continuous presence of IMP CD4+ T cells, and we found this protocol fully prevented the wasting in MHCII−/−→WT chimeras (Fig. 2G).

DCs are the major expressers of MHCII among hematopoietic cells and enforce clonal deletion of self-reactive T cells, as well as the induction of Tregs (26). It is possible that DC-MHCII contributes to the development of IMP CD4+ T cells, which are protective in syngeneic bone marrow chimeras. We therefore used an MHCII transgenic model where MHCII is re-expressed in MHCII−/− mice solely on CD11c+ DCs (Tg(CD11c-MHCII)/MHCII−/−, referred to as MHCIIDC hereafter) (17) as donors to investigate whether DC-restricted MHCII is important for the development of IMP CD4+ T cells and prevention of disease following BMT. We found that MHCII expression by DCs did not induce more IMP-like CD4+ T cells compared with complete MHCII deficiency; however, it led to “licensing” of functional IMP CD4+ T cells to become potent IFN-γ producers (Fig. 3A, 3B). Furthermore, transfer of MHCIIDC bone marrow to WT mice (MHCIIDC→WT) completely rescues IMP CD4+ T cell functional development (Fig. 3C, 3D) and prevents wasting (Fig. 3E). Additionally, despite some variation in T cell–derived TNF-α by cells from the MHCIIDC→WT chimeras, there was significantly enhanced IL-10 production (Fig. 3F). These data indicate an important role for DC-MHCII in promoting regulatory IMP CD4+ T cell development and preventing pathogenesis in syngeneic bone marrow transplantation.

FIGURE 3.

DC-restricted expression of MHCII is sufficient to rescue IMP CD4+ T cells and suppress development of disease. (A and B) Representative plots and summary of expression of CD44, CD62L, and IFN-γ by CD4+ T cells (percentages [left] and numbers [middle] of IMP CD4+ T cells and percentage of CD44hi IFN-γ–secreting CD4+ T cells [right]) in indicated mice. (C and D) Bone marrow chimeras were generated as indicated, and CD4+ T cells of donor origin were analyzed. (C) Representative plots and (D) summary of expression of CD44, CD62L, and IFN-γ by CD4+ T cells of donor origin (percentages [left] and numbers [middle] of IMP CD4+ T cells and percentage of CD44hi IFN-γ–secreting CD4+ T cells [right]) in indicated chimeras. (E) DC-restricted expression of MHCII on donor bone marrow prevents weight loss; n = 5 shown. (F) IL-10 and TNF-α secreted by anti-CD3/CD28–stimulated splenocytes from the indicated chimeric mice. All data represent or were combined from results of at least two independent experiments. The p values in (B), (D), and (F) were determined by Student t test, and in (E) by a two-way ANOVA.

FIGURE 3.

DC-restricted expression of MHCII is sufficient to rescue IMP CD4+ T cells and suppress development of disease. (A and B) Representative plots and summary of expression of CD44, CD62L, and IFN-γ by CD4+ T cells (percentages [left] and numbers [middle] of IMP CD4+ T cells and percentage of CD44hi IFN-γ–secreting CD4+ T cells [right]) in indicated mice. (C and D) Bone marrow chimeras were generated as indicated, and CD4+ T cells of donor origin were analyzed. (C) Representative plots and (D) summary of expression of CD44, CD62L, and IFN-γ by CD4+ T cells of donor origin (percentages [left] and numbers [middle] of IMP CD4+ T cells and percentage of CD44hi IFN-γ–secreting CD4+ T cells [right]) in indicated chimeras. (E) DC-restricted expression of MHCII on donor bone marrow prevents weight loss; n = 5 shown. (F) IL-10 and TNF-α secreted by anti-CD3/CD28–stimulated splenocytes from the indicated chimeric mice. All data represent or were combined from results of at least two independent experiments. The p values in (B), (D), and (F) were determined by Student t test, and in (E) by a two-way ANOVA.

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We have also tested the involvement of B cells that also express MHCII, along with the costimulatory ligands CD80/86 in the functional development of IMP CD4+ T cells. We found that although the lack of B cells (in μMT mice) significantly reduces the total T cell number and thus IMP CD4+ T cell number, it does not affect the percentage of IMP CD4+ T cells (Fig. 4A, 4B). In contrast, CD80/86 seems to be important for both number and function of IMP CD4+ T cells (Fig. 4A, 4B), and bone marrow chimeras from WT or CD80/86 DKO mice in MHCII−/− recipients revealed that hematopoietic expression of CD80/86 expands IMP CD4+ T cells, but is dispensable for their functional maturation (Fig. 4C, 4D). Thus, DC-MHCII in the hematopoietic system is the dominant factor for functional development of IMP CD4+ T cells, whereas B cells and hematopoietic CD80/86 regulate the population size of these cells.

FIGURE 4.

B cells and CD80/86 coactivators regulate the dynamics of IMP CD4+ T cell development. Splenocytes from indicated mice were analyzed. Bone marrow chimeras were generated as indicated and donor CD4+ T cells were analyzed. (A and C) Representative plots of expression of CD44, CD62L, and IFN-γ by CD4+ T cells. (B and D) Total numbers of CD4+ T cells (upper left), percentages (upper right), and numbers (lower left) of IMP CD4+ T cells and percentages of CD44hi IFN-γ–secreting CD4+ T cells (lower right) are shown. All data represent or were combined from results of two independent experiments. The p values were determined by Student t test.

FIGURE 4.

B cells and CD80/86 coactivators regulate the dynamics of IMP CD4+ T cell development. Splenocytes from indicated mice were analyzed. Bone marrow chimeras were generated as indicated and donor CD4+ T cells were analyzed. (A and C) Representative plots of expression of CD44, CD62L, and IFN-γ by CD4+ T cells. (B and D) Total numbers of CD4+ T cells (upper left), percentages (upper right), and numbers (lower left) of IMP CD4+ T cells and percentages of CD44hi IFN-γ–secreting CD4+ T cells (lower right) are shown. All data represent or were combined from results of two independent experiments. The p values were determined by Student t test.

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In the absence of the non–receptor tyrosine kinase Itk, the percentage of functional (ability to produce IFN-γ) IMP CD4+ T cells are significantly increased (4) (Fig. 5A), and the combined absence of Itk and MHCII also surprisingly leads to significantly increased numbers of IMP CD4+ T cells, and these cells now have the capability to produce IFN-γ, unlike those seen in the MHCII−/− (Fig. 5A, 5B). Furthermore, Itk was dispensable for IMP CD4+ T cell development through hematopoietic MHCII selection (Itk−/−→MHCII−/− chimeras, Supplemental Fig. 2). This suggests that Itk plays a negative role in the development or licensing of IMP CD4+ T cells. We used the Itk−/−MHCII−/− mice as bone marrow donors to determine the role of Itk in the development and function of IMP CD4+ T cells, as well as on the development of disease. We found that the absence of Itk in MHCII−/− bone marrow rescued the development of IMP CD4+ T cells (Fig. 5C, 5D). Additionally, the weight loss observed in the MHCII−/−→WT mice is significantly attenuated in the WT recipients of Itk−/−MHCII−/− bone marrow (Itk−/−MHCII−/−→WT), and compared with WT→WT control, this weight loss is delayed (Fig. 5E). Note that Itk deficiency also restored the ability of splenic T cells to produce IL-10 (Fig. 5F). Thus, Itk plays a regulatory role in the development and function of IMP CD4+ T cells after syngeneic BMT.

FIGURE 5.

The absence of Itk rescues IMP CD4+ T cell development and partially suppresses wasting. (A and B) Representative plots and summary of expression of CD44, CD62L, and IFN-γ by CD4+ T cells (percentages [left] and numbers [middle] of IMP CD4+ T cells and percentage of CD44hi IFN-γ–secreting CD4+ T cells [right]) in indicated mice. (C and D) Bone marrow chimeras were generated as indicated, and CD4+ T cells of donor origin were analyzed. (C) Representative plots and (D) summary of expression of CD44, CD62L, and IFN-γ by CD4+ T cells of donor origin (percentages [left] and numbers [middle] of IMP CD4+ T cells and percentage of CD44hi IFN-γ–secreting CD4+ T cells [right]) in indicated chimeras are shown. (E) Absence of Itk in MHCII−/− bone marrow reduced severity of body weight loss of WT recipients; n = 5 shown. (F) IL-10 and TNF-α secreted by anti-CD3/CD28–stimulated splenocytes from the indicated chimeric mice. All data represent or were combined from results of at least two independent experiments. The p values in (B), (D), and (F) were determined by Student t test, and in (E) by a two-way ANOVA.

FIGURE 5.

The absence of Itk rescues IMP CD4+ T cell development and partially suppresses wasting. (A and B) Representative plots and summary of expression of CD44, CD62L, and IFN-γ by CD4+ T cells (percentages [left] and numbers [middle] of IMP CD4+ T cells and percentage of CD44hi IFN-γ–secreting CD4+ T cells [right]) in indicated mice. (C and D) Bone marrow chimeras were generated as indicated, and CD4+ T cells of donor origin were analyzed. (C) Representative plots and (D) summary of expression of CD44, CD62L, and IFN-γ by CD4+ T cells of donor origin (percentages [left] and numbers [middle] of IMP CD4+ T cells and percentage of CD44hi IFN-γ–secreting CD4+ T cells [right]) in indicated chimeras are shown. (E) Absence of Itk in MHCII−/− bone marrow reduced severity of body weight loss of WT recipients; n = 5 shown. (F) IL-10 and TNF-α secreted by anti-CD3/CD28–stimulated splenocytes from the indicated chimeric mice. All data represent or were combined from results of at least two independent experiments. The p values in (B), (D), and (F) were determined by Student t test, and in (E) by a two-way ANOVA.

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BMT with MHCII-deficient murine bone marrow results in severe wasting accompanied with lethal colitis in syngeneic WT recipients (3). The cellular mechanism of this disease is not yet elucidated. Nevertheless, this syngeneic MHCII−/−→WT transplant-induced pathogenesis provides an excellent model to investigate factors involved in the failure of “perfect-match” bone marrow grafts. Onset of disease may be due to reduced regulatory mechanisms in the periphery (3), or unfettered autoreactive T cells caused by failure of hematopoietic MHCII–mediated thymic negative selection (2). The latter mechanism has been proposed because although WT→MHCII−/− chimeras fail to generate normal numbers of conventional Tregs (27), they do not appear sick. Furthermore, thymectomy of WT recipients, which eliminates positive selection, prevented pathogenesis induced by transplant of MHCII−/− bone marrow (28). However, these findings do not fully support the explanation that the pathogenesis in MHCII−/−→WT chimeras is due to the lack of negative selection, and it is likely that this is coupled with a lack of regulatory system. Our data support the view that reduced development of the regulatory compartment significantly contributes to the development of wasting syndrome in MHCII−/−→WT BMTs, and that the full spectrum of IMP CD4+ T cells is requisite in this regulatory system. Although the systems are fairly different, in allogeneic BMT, donor naive but not CD44hiCD62Llo IMP CD4+ T cells were responsible for the onset of GVHD (29), and chimeras with comparable numbers of Tregs to healthy control chimeras still developed severe chronic GVHD (30). Additionally, whereas as few as 104 CD4+ invariant NKT cells have been shown to be able to suppress acute GVHD during allogeneic bone marrow transplantation (31), we found no difference in invariant NKT cell number between WT→MHCII−/− (healthy) and MHCII−/−→WT (sick) chimeras, which both have significantly lower numbers of invariant NKT cells compared with WT→WT controls (Supplemental Fig. 4C). These findings support our conclusion that the reduction of invariant NKT cells may not be the reason of pathogenesis in MHCII−/−→WT chimeras. These might implicate, in general, a regulatory system provided by the memory-like CD4+ T cells.

Bone marrow–derived DCs play critical roles in the development of self-tolerance via negative selection in the thymus and the development of Foxp3+ Tregs (3235). Ablation of DCs in donor marrow leads to CD4+ T cell infiltration into multiple peripheral organs in syngeneic WT recipients (36), and bone marrow with DCs that are defective in cognate CD4+ T cell recognition resulted in impaired peripheral tolerance, with fatal CD8+ T cell cytotoxicity in syngeneic WT recipients (37). In this study, we identified DCs as the primary hematopoietic cells expressing MHCII required for selection and/or licensing of functional IMP CD4+ T cells. We use the term “licensing” in this case because MHCII−/− mice have cells of the innate memory phenotype, but they do not produce IFN-γ upon stimulation. In contrast, expression of MHCII only on CD11c+ DCs rescues the function (IFN-γ production) without affecting the numbers of these cells (cf. Fig. 3B). Based on similar use of the word in NK cell biology (38), we refer to the IMP CD4 cells that develop under MHC null condition as “licensed” for cytokine production by expression of MHCII on CD11c+ DCs. Previous analysis of the effect of expressing MHCII on CD4+ T cells showed that these cells can contribute to selection of other CD4+ T cells with a similar memory-like phenotype, defined as thymocyte-selected CD4+ T cells (i.e., CD44hi) (5, 39). However, it is difficult to rule out that the CD4 promoter/enhancer used to express MHCII in these studies does not also result in expression of MHCII on at least a subset of DCs, because DCs have been reported to also express CD4 (40). The properties of these cells in BMT have not been evaluated, and so it is not clear whether these cells are the same population of IMP CD4+ T cells described in the present study. Nevertheless, we showed that DC expression of MHCII is requisite and sufficient for IMP CD4+ T cell development, T cell–derived IL-10 production, and prevention of wasting in MHCII−/−→WT BMT model. Our data suggest that DCs may function to license CD4+ T cells with an innate regulatory program, allowing them to suppress T cell–mediated pathogenesis in syngeneic bone marrow graft.

When Itk is not expressed in MHCII−/− donor bone marrow, development of IMP CD4+ T cells is rescued, along with partial suppression of wasting. It might be paradoxical that Itk regulates CD4+ TCR signaling via MHCII, whereas Itk−/−MHCII−/− bone marrow exhibited better differentiation of IMP CD4+ T cells. One possible explanation is that the IMP CD4+ T cells require weak TcR signaling to expand the population and to achieve functional maturation, and the absence of Itk weakens TcR signals such that these cells develop independently of hematopoietic MHCII. Whether these speculations are correct requires further investigation; nevertheless, targeting Itk may potentially serve as an in vivo supplement to generate IMP CD4+ T cells, or to enrich regulatory IMP CD4+ T cells in syngeneic bone marrow grafts.

We thank Shailaja Hegde, Nicole Bem, Susan Magargee, Lavanya G. Sayam, Gabriel Balmus, Jennifer D. Mosher, Yuting Bai, Tina Chew, Dr. Rod Getchell, and Dr. Walter Iddings for technical assistance. We also thank Margaret Potter and Amie Wood for animal care.

This work was supported by National Institutes of Health Grants AI051626 and AI065566 (to A.A.).

The microarray data presented in this article have been submitted to the National Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/gds) under accession number GSE46892.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BMT

bone marrow transplantation

DC

dendritic cell

GVHD

graft-versus-host disease

IMP

innate memory phenotype

Itk

IL-2–inducible T cell kinase

MHCII

MHC class II

Treg

regulatory T cell

WT

wild-type.

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The authors have no financial conflicts of interest.

Supplementary data