T cell-induced inflammation of the small and large intestine in immunodeficient mice

It is well known that transfer of CD4+CD45RBhigh (na?ve) T cells into syngeneic lymphocyte-deficient mice induces chronic colitis. However, no studies have reported the presence of small bowel inflammation in this T cell-dependent model. Therefore, the objective of this study was to evaluate and compare small and large bowel inflammation induced by transfer of na?ve T cells into two different immunodeficient recipient mice. T and B cell-deficient recombinase activating gene 1-deficient [RAG knockout (KO)] and T cell-deficient T cell receptor-beta x T cell receptor-delta double-deficient (TCR KO) mice were reconstituted with wild-type na?ve T cells and observed for signs of disease. We found that reconstituted RAG KO mice developed moderate to severe colitis and inflammation of the entire small intestine at 6-8 wk after T cell transfer. Adoptive transfer of na?ve T cells into TCR KO mice induced a milder form of chronic colitis and small bowel inflammation that was confined primarily to the duodenum at 10-12 wk after T cell transfer. T helper cell 1 and macrophage-derived proinflammatory cytokine mRNA levels correlated well with the localization and severity of the chronic large and small bowel inflammation. In addition, we observed comparable homing and expansion of donor lymphocytes in the gut and secondary lymphoid tissues of both recipients. Taken together, our data demonstrate that transfer of na?ve T cells into immunodeficient recipient mice induces both chronic small and large bowel inflammation and that the presence of B cells in the TCR KO recipients may play a role in regulating chronic intestinal inflammation.     

TCR-mediated involvement of CD4+ transgenic T cells in spontaneous inflammatory bowel disease in lymphopenic mice.

Spontaneous colitis resembling ulcerative colitis developed in 3 of 10 independent TCR transgenic (Tg) mouse lines maintained under specific pathogen-free conditions. All three susceptible lines were CD4 lymphopenic, whereas resistant lines had normal numbers of CD4+ T cells. Thus, cytochrome c-specific 5C.C7 TCR Tg mice developed colitis only when crossed onto a SCID- or Rag-1-deficient background. A second line of lymphopenic cytochrome c-specific Tg mice bearing the AND TCR also developed colitis. In both cases, CD4+ T cells expressing the Tg-encoded TCR were preferentially activated in inflamed colons compared with lymph nodes or spleens. In contrast, Tg+CD4+ T cells remained quiescent in both inflamed and unaffected colons in another line of susceptible Tg mice carrying a TCR specific for myelin basic protein, suggesting a fortuitous cross-reactivity of the IEk-restricted cytochrome c-reactive AND and 5C.C7 TCRs with an Ag present in the gut. The percentage of CD4+ T cells expressing only endogenous TCR alpha-chains was increased consistently in inflamed colons in AND as well as 5C.C7 Rag-1-/- TCR Tg mice, suggesting that polyclonal CD4+ T cells were also involved in the pathogenesis of spontaneous colitis. Moreover, our data indicate that some alpha-chain rearrangement was still occurring in TCR Tg mice on a Rag-1-/- background, since activated CD4+ T cells expressing endogenously rearranged alpha-chains paired with the Tg-encoded beta-chain were detected consistently in the colons of such mice.     

Regulation of lymphoid homeostasis by IL-2 receptor signals in vivo

High-affinity IL-2R signals are required for peripheral lymphoid homeostasis in vivo. We found that CD25 was required for regulation of peripheral T cells in mice bearing either the DO11.10 MHC class II-restricted TCR transgene or an Iabeta-null mutation, suggesting that MHC class I- and class II-dependent T cell subsets are regulated independently by IL-2R signals. In contrast, deregulation of serum IgG1 levels in CD25-/- mice was dependent on CD4+ T cells. T cell expansion in DO11.10 CD25-/- mice was not preferential for cells escaping allelic exclusion by the TCR transgene, but was suppressed by a Rag-2-null mutation. Together, these findings suggest that endogenous TCR are required to trigger T cell expansion, but that CD25 regulates T cells activated by low-specificity signals. Expansion of DO11.10 T cells in response to cognate Ag was modestly reduced in CD25-/- T cells transferred into the normal lymphoid compartments of BALB/c mice. Moreover, activation-induced clonal contraction and apoptosis in vivo were intact in the absence of CD25. These data indicate that the regulatory role of high-affinity IL-2R signals extends beyond the control of Ag-specific responses and suggest a role for these signals in control of bystander T cell activation.     

TGF-beta 1 regulates antigen-specific CD4+ T cell responses in the periphery

T cell expansion typically is due to cognate interactions with specific Ag, although T cells can be experimentally activated through bystander mechanisms not involving specific Ag. TGF-beta1 knockout mice exhibit a striking expansion of CD4+ T cells in the liver by 11 days of age, accompanied by CD4+T cell-dependent necroinflammatory liver disease. To examine whether hepatic CD4+T cell expansion in TGF-beta1(-/-) mice is due to cognate TCR-peptide interactions, we used spectratype analysis to examine the diversity in TCR Vbeta repertoires in peripheral CD4+T cells. We reasoned that Ag-nonspecific T cell responses would yield spectratype profiles similar to those derived from control polyclonal T cell populations, whereas Ag-specific T cell responses would yield perturbed spectratype profiles. Spleen and liver CD4+T cells from 11-day-old TGF-beta1(-/-) mice characteristically exhibited highly perturbed nonpolyclonal distributions of TCR Vbeta CDR3 lengths, indicative of Ag-driven T cell responses. We quantitatively assessed spectratype perturbation to derive a spectratype complexity score. Spectratype complexity scores were considerably higher for TGF-beta1(-/-) CD4+ T cells than for TGF-beta1(+/-) CD4+T cells. TCR repertoire perturbations were apparent as early as postnatal day 3 and preceded both hepatic T cell expansion and liver damage. By contrast, TGF-beta1(-/-) CD4+ single-positive thymocytes from 11-day-old mice exhibited normal unbiased spectratype profiles. These results indicate that CD4+ T cells in TGF-beta1(-/-) mice are activated by and respond to self-Ags present in the periphery, and define a key role for TGF-beta1 in the peripheral regulation of Ag-specific CD4+ T cell responses.     

Macrophages driven to a novel state of activation have anti-inflammatory properties in mice

Recurrent episodes of inflammation underlie numerous pathologies, notably those of inflammatory bowel diseases. In this study, we describe a population of macrophages in a novel state of activation that mitigates colitis in mice. The cells responsible for this effect, called IFN-gamma-stimulated monocyte-derived cells (IFNgamma-MdC), derive from mouse spleen, blood, and bone marrow monocytes and are distinguished from known macrophage populations by mode of generation, cell surface phenotype, and function. IFNgamma-MdC only arise when macrophages are cultivated in the presence of CD40L-expressing CD4+ T cells, M-CSF, and IFN-gamma. IFNgamma-MdC express markers including F4/80, CD11b/c, CD86, and CD274; they are negative for CD4, CD8, Gr1, CD19, CD80, and CD207. Functionally, IFNgamma-MdC are defined by their capacity to enrich cocultured T cell populations for CD4+CD25+Foxp3+ regulatory cells; this enrichment, constituting up to 60% or more of residual lymphocytes, is attributed to an expansion, but also to a cell contact and caspase-dependent depletion of activated T cells. In mice, IFNgamma-MdC delivered i.v. traffic to gut-associated peripheral lymphoid tissues, including the mesenteric lymph nodes, Peyer's patches, and colonic mucosa, and promote the clinical and histological resolution of chronic colitis. We conclude that IFNgamma-MdC represent macrophages in a novel state of activation, possessing multiple T cell-suppressive effects with therapeutic potential for the treatment of autoimmune inflammation.     

Requirement for CD4+ T cells in V beta 4+CD8+ T cell activation associated with latent murine gammaherpesvirus infection.

A CD8+ T cell lymphocytosis in the peripheral blood is associated with the establishment of latency following intranasal infection with murine gammaherpesvirus-68. Remarkably, a large percentage of the activated CD8+ T cells of mice expressing different MHC haplotypes express V beta 4+ TCR. Identification of the ligand driving the V beta 4+CD8+ T cell activation remains elusive, but there is a general correlation between V beta 4+CD8+ T cell stimulatory activity and establishment of latency in the spleen. In the current study, the role of CD4+ T cells in the V beta 4+CD8+ T cell expansion has been addressed. The results show that CD4+ T cells are essential for expansion of the V beta 4+CD8+ subset, but not other V beta subsets, in the peripheral blood. CD4+ T cells are required relatively late in the antiviral response, between 7 and 11 days after infection, and mediate their effect independently of IFN-gamma. Assessment of V beta 4+CD8+ T cell stimulatory activity using murine gammaherpesvirus-68-specific T cell hybridomas generated from latently infected mice supports the idea that CD4+ T cells control levels of the stimulatory ligand that drives the V beta 4+CD8+ T cells. As V beta 4+CD8+ T cell expansion also correlates with levels of activated B cells, these data raise the possibility that CD4+ T cell-mediated B cell activation is required for optimal expression of the stimulatory ligand. In addition, in cases of low ligand expression, there may also be a direct role for CD4+ T cell-mediated help for V beta 4+CD8+ T cells.     

TNF-alpha controls intrahepatic T cell apoptosis and peripheral T cell numbers

At the end of an immune response, activated lymphocyte populations contract, leaving only a small memory population. The deletion of CD8(+) T cells from the periphery is associated with an accumulation of CD8(+) T cells in the liver, resulting in both CD8(+) T cell apoptosis and liver damage. After adoptive transfer and in vivo activation of TCR transgenic CD8(+) T cells, an increased number of activated CD8(+) T cells was observed in the lymph nodes, spleen, and liver of mice treated with anti-TNF-alpha. However, caspase activity was decreased only in CD8(+) T cells in the liver, not in those in the lymphoid organs. These results indicate that TNF-alpha is responsible for inducing apoptosis in the liver and suggest that CD8(+) T cells escaping this mechanism of deletion can recirculate into the periphery.     

Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific

CD4+CD25+ T cells represent a unique population of "professional" suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+CD25- cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+CD25- Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+CD25- T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this T-T interaction remain to be characterized.     

Cutting edge: two distinct mechanisms lead to impaired T cell homeostasis in Janus kinase 3- and CTLA-4-deficient mice

Cytokine receptor signaling and costimulatory receptor signaling play distinct roles in T cell activation. Nonetheless, deficiencies in either of these pathways lead to seemingly similar phenotypes of impaired T cell homeostasis. A dramatic expansion of CD4(+) peripheral T cells with an activated phenotype has been observed in both Janus kinase (Jak) 3-deficient and CTLA-4-deficient mice. Despite these similarities, the mechanisms driving T cell expansion may be distinct. To address this possibility, we examined the TCR repertoire of peripheral T cells in Jak3(-/-) and CTLA-4(-/-) mice using complementarity-determining region 3 spectratype analysis. Interestingly, a restricted and highly biased TCR repertoire was observed in the Jak3(-/-) T cells, strongly supporting a role for foreign Ag in the activation and expansion of these cells. In contrast, CTLA-4(-/-) T cells had a diverse and unbiased TCR repertoire, suggestive of a universal, Ag-independent mechanism of activation and expansion. These findings provide insight into the diverse mechanisms controlling T cell homeostasis.     

Intestinal intraepithelial TCRγδ+ T cells are activated by normal commensal bacteria

TCR???⁺ T cells play a critical role in protecting the intestinal mucosa against pathogenic infection. In the absence of infection, TCR???⁺ T cell activation must be continuously regulated by T regulatory cells (Treg) to prevent the development of colitis. However, the activation of intestinal TCR???⁺ T cells under normal conditions has not been clearly resolved. In order to determine TCR???⁺ T cell activation in vivo, we designed an NF-??B based reporter system. Using the recombinant lentiviral method, we delivered the NF-??B reporter to isolated TCR???⁺ T cells, which were then adoptively transferred into normal mice. Our data indicate that the NF-??B activation level in TCR???⁺ T cells is higher in the intestinal intraepithelial layer than in the lamina propria region. In addition, the surface expression level of lymphocyte activation marker CD69 in TCR???⁺ T cells is also higher in the intestinal intraepithelial layer and this activation was reduced by Sulfatrim treatment which removes of commensal bacteria. Collectively, our data indicate that the TCR???⁺ T cell population attached to the intestinal lumen is constitutively activated even by normal commensal bacteria.     

Nod2 Activates NF-kB in CD4+ T Cells but Its Expression Is Dispensable for T Cell-Induced Colitis

Although the etiology of Crohn''s disease (CD) remains elusive this disease is characterized by T cell activation that leads to chronic inflammation and mucosal damage. A potential role for maladaptation between the intestinal microbiota and the mucosal immune response is suggested by the fact that mutations in the pattern recognition receptor Nod2 are associated with higher risks for developing CD. Although Nod2 deletion in CD4⁺ T cells has been shown to impair the induction of colitis in the murine T cell transfer model, the analysis of T cell intrinsic Nod2 function in T cell differentiation and T cell-mediated immunity is inconsistent between several studies. In addition, the role of T cell intrinsic Nod2 in regulatory T cell (Treg) development and function during colitis remain to be analyzed. In this study, we show that Nod2 expression is higher in activated/memory CD4⁺ T cells and its expression was inducible after T cell receptor (TCR) ligation. Nod2 stimulation with muramyl dipeptide (MDP) led to a nuclear accumulation of c-Rel NF-kB subunit. Although functionally active in CD4⁺ T cells, the deletion of Nod2 did not impair the induction and the prevention of colitis in the T cell transfer model. Moreover, Nod2 deletion did not affect the development of Foxp3⁺ Treg cells in the spleen of recipient mice and Nod2 deficient CD4 T cells expressing the OVA specific transgenic TCR were able to differentiate in Foxp3⁺ Treg cells after OVA feeding. In vitro, CD25⁺ Nod2 deficient T cells suppressed T cell proliferation as well as wild type counter parts and T cell stimulation with MDP did not affect the proliferation and the cytokine secretion of T cells. In conclusion, our data indicate that Nod2 is functional in murine CD4⁺ T cells but its expression is dispensable for the T cell regulation of colitis.     

Abnormal thymocyte development and production of autoreactive T cells in T cell receptor transgenic autoimmune mice.

Development of a C57BL/6-+/+ TCR transgenic mouse containing the rearranged TCR alpha- and beta-chain specific for the Db + HY male Ag results in production of a nearly monoclonal population of early thymocytes expressing the Db + HY reactive TCR. These thymocytes are autoreactive in H-2Db male mice and undergo clonal deletion and down-regulation of CD8. To study the effect of the lpr gene on development of autoreactive T cells, these transgenic mice were backcrossed with C57BL/6-lpr/lpr mice. T cell populations in the thymus and spleen were analyzed by three-color flow cytometry for expression of CD4, CD8, and TCR. The thymus of TCR transgenic H-2b/b lpr/lpr male mice had an increase in percent and absolute number of CD8dull thymocytes compared to TCR transgenic H-2b/b +/+ male mice. However, there was not a complete defect in clonal deletion, because clonal deletion and down-regulation of CD8 was apparent in both +/+ and lpr/lpr H-2Db HY+ male mice compared to H-2Db HY- female mice. The phenotype of splenic T cells was almost identical in TCR transgenic +/+ and lpr/lpr males with about 50% CD4-CD8- T cells and 50% CD8+ T cells. However, there was a dramatic increase in the SMLR proliferative response of splenic T cells from TCR transgenic lpr/lpr males compared to TCR transgenic +/+ males. To determine the specificity of this response, spleen cells from TCR transgenic lpr/lpr and +/+ mice were cultured with irradiated H-2b/b and H-2k/k male and female spleen cells. T cells from TCR transgenic C57BL/6-lpr/lpr male mice had an increased proliferative response to H-2b/b male spleen cells compared to T cells from TCR transgenic C57BL/6(-)+/+ male mice, but both lpr/lpr and +/+ mice had a minimal response to irradiated H-2b/b female or H-2k/k male or female stimulator cells. The splenic T cells from TCR transgenic lpr/lpr mice also had an increased specific cytotoxic activity against H-2b/b male target cells compared to TCR transgenic +/+ mice. These results demonstrate that there is a defect in negative selection of self-reactive T cells in the thymus of lpr/lpr mice and a defect in induction or maintenance of clonal anergy of self-reactive T cells in the periphery of lpr/lpr mice.     

Effect of age on homeostasis of lymphocytes in an interleukin-7-deficient mouse model

Interleukin (IL)-7 is thought to be a non-redundant cytokine for lymphopoiesis as there is a reduction of T and B cells in peripheral blood (PB) and a loss of TCRγδ+ cells in PB and bone marrow (BM) in IL-7?/? mice. To investigate whether the absence of IL-7 influences the organ-dependent distribution of the lymphocytes, we analyzed single cell suspensions of several organs (BM, lung, liver, small intestine, and spleen) at different ages (three and 12 months) of IL-7+/+ and IL-7?/? mice using flow cytometry; immunohistochemical staining was performed on frozen sections of various organs. We observed lymphocytopenia in almost all organs of IL-7?/? mice, but normal counts in the liver and the lung of three-month-old IL-7?/? mice. CD4+ and CD8+ cell numbers were decreased in the spleen and the BM. With aging, we found a greater increase in CD4+ and CD8+ cells in the BM of IL-7?/? than in IL-7+/+ mice, particularly of memory cells. The spleen of IL-7?/? mice was characterized by lymphocytopenia. We challenge the view that IL-7 is a non-redundant cytokine for lymphocyte development. Some of the changes observed, e.g. partial absence of TCRγδ+ T cells in the PB, BM and small intestine and complete loss in liver, lung and spleen, may be due to the altered organ distribution instead of a defect in γδ+ T cell lymphopoiesis. In this model, aging leads to a significantly altered composition of lymphocyte subsets, and the lack of IL-7 seems to accelerate this process.     

Cutting edge: Generation of colitogenic Th17 CD4 T cells is enhanced by IL-17+ γδ T cells

Th 17 cells have been implicated in the pathogenesis of colitis; however, a cellular mechanism by which colitogenic Th17 immunity arises in vivo remains unclear. In this study, we report that a subset of IL-17(+) γδ T cells plays a crucial role in enhancing in vivo Th17 differentiation and T cell-mediated colitis. TCRβ(-/-) mice were highly susceptible to T cell-mediated colitis, whereas TCRβδ(-/-) mice were resistant to the disease. Importantly, cotransfer of IL-17(+) but not of IL-17(-) γδ T cells with CD4 T cells was sufficient to enhance Th17 differentiation and induce full-blown colitis in TCRβδ(-/-) recipients. Collectively, our results provide a novel function of IL-17(+) γδ T cell subsets in supporting in vivo Th17 differentiation and possibly in fostering the development of intestinal inflammation.     

T cell activation by coxsackievirus B4 antigens in type 1 diabetes mellitus: evidence for selective TCR Vbeta usage without superantigenic activity.

Numerous clinical and epidemiological studies link enteroviruses such as the Coxsackie virus group with the autoimmune disease type 1 diabetes mellitus (DM). In addition, there are reports that patients with type 1 DM are characterized by skewing of TCR Vbeta chain selection among peripheral blood and intraislet T lymphocytes. To examine these issues, we analyzed TCR Vbeta chain-specific up-regulation of the early T cell activation marker, CD69, on CD4 T cells after incubation with Coxsackievirus B4 (CVB4) Ags. CD4 T cells bearing the Vbeta chains 2, 7, and 8 were the most frequently activated by CVB4. Up-regulation of CD69 by different TCR families was significantly more frequent in new onset type 1 DM patients (p = 0.04), 100% of whom (n = 8) showed activation of CD4 T cells bearing Vbeta8, compared with 50% of control subjects (n = 8; p = 0.04). T cell proliferation after incubation with CVB4 Ags required live, nonfixed APCs, suggesting that the selective expansion of CD4 T cells with particular Vbeta chains resulted from conventional antigen processing and presentation rather than superantigen activity. Heteroduplex analysis of TCR Vbeta chain usage after CVB4 stimulation indicated a relatively polyclonal, rather than oligo- or monoclonal response to viral Ags. These results provide evidence that new-onset patients with type 1 DM and healthy controls are primed against CVB4, and that CD4 T cell responses to the virus have a selective TCR Vbeta chain usage which is driven by viral Ags rather than a superantigen.     

Mechanisms of Vdelta1 gammadelta T cell activation by microbial components

There are two major subsets of gammadelta T cell in humans. Vgamma2Vdelta2 T cells predominate in the circulation and significantly expand in vivo during a variety of infectious diseases. Ags identified for the Vdelta2 T cells are nonpeptide phosphate, amine, and aminobisphosphonate compounds. In contrast, Vdelta1-encoded TCRs account for the vast majority of gammadelta T cells in tissues such as intestine and spleen. Some of these T cells recognize CD1c and MHC class I-related chain (MICA/B) molecules [correction]. These T cells are cytotoxic and use both perforin- and Fas-mediated cytotoxicity. A fundamental question is how these gammadelta T cells are activated during microbial exposure to carry out effector functions. In this study, we provide evidence for a mechanism by which Vdelta1 gammadelta T cells are activated by inflammatory cytokines in the context of the Vdelta1 TCR. Dendritic cells are necessary as accessory cells for microbial Ag-mediated Vdelta1 gammadelta T cell activation. Cytokine (IL-12), adhesion (LFA3/CD2, LFA1/ICAM1) and costimulatory (MHC class I-related chain (MICA/B) molecules/NK-activating receptor G2D) molecules play a significant role along with Vdelta1 TCR in this activation.     

Peroxisome proliferator-activated receptor gamma is required for regulatory CD4+ T cell-mediated protection against colitis

Peroxisome proliferator-activated receptor (PPAR) gamma activation has been implicated in the prevention of immunoinflammatory disorders; however, the mechanisms of regulation of effector and regulatory CD4+ T cell functions by endogenously activated PPAR-gamma remain unclear. We have used PPAR-gamma-deficient CD4+ T cells obtained from tissue-specific PPAR-gamma null mice (i.e., PPAR-gamma fl/fl; MMTV-Cre+) to investigate the role of endogenous PPAR-gamma on regulatory T cell (Treg) and effector CD4+ T cell function. Overall, we show that the loss of PPAR-gamma results in enhanced Ag-specific proliferation and overproduction of IFN-gamma in response to IL-12. These findings correlate in vivo with enhanced susceptibility of tissue-specific PPAR-gamma null mice to trinitrobenzene sulfonic acid-induced colitis. Furthermore, the transfer of purified PPAR-gamma null CD4+ T cells into SCID recipients results in enteric disease. To test the assertion that the deficiency of PPAR-gamma in Treg impairs their ability to prevent effector T cell-induced colitis, we performed cotransfer studies. These studies demonstrate that PPAR-gamma-expressing, but not PPAR-gamma null Treg, prevent colitis induced by transfer of naive CD4+ T cells into SCID recipients. In line with these findings, the production of IFN-gamma by spleen and mesenteric lymph node-derived CD4+ T cells was down-regulated following transfer of PPAR-gamma-expressing, but not PPAR-gamma null, Treg. In conclusion, our data suggest that endogenous PPAR-gamma activation represents a Treg intrinsic mechanism of down-regulation of effector CD4+ T cell function and prevention of colitis.