Rita Fucs

Frequency of Natural Regulatory CD4+CD25+ T Lymphocytes Determines the Outcome of Tolerance across Fully Mismatched MHC Barrier through Linked Recognition of Self and Allogeneic Stimuli

We show in this study that long-term tolerance to allogeneic skin grafts can be established in the absence of immunosuppression by the combination of the following elements: 1) augmenting the frequency of regulatory CD4+CD25+ T cells (Treg) and 2) presentation of the allogeneic stimuli through linked recognition of allo- and self-epitopes on semiallogeneic F1 APCs. BALB/c spleen cells enriched for CD4+CD25+ T lymphocytes were transferred either to BALB/c nu/nu mice or to BALB/c nu/nu previously injected with F1(BALB/c × B6.Ba) spleen cells, or else grafted with F1(BALB/c × B6.Ba) skin (chimeric BALB/c nu/nu-F1). Chimeric BALB/c nu/nu-F1 reconstituted with syngeneic CD25+-enriched spleen cells were unable to reject the previously transferred F1(BALB/c × B6.Ba) spleen cells or F1(BALB/c × B6.Ba) skin grafts, and a specific tolerance to a secondary B6 graft was obtained, with rejection of third-party CBA grafts. BALB/c nu/nu mice reconstituted only with syngeneic CD25+-enriched spleen cells rejected both B6 and CBA skin grafts. In contrast, when chimeric BALB/c nu/nu-F1 were reconstituted with spleen populations comprising normal frequencies of Treg cells, the linked recognition of allo and self resulted in breaking of self tolerance and rejection of syngeneic grafts, strongly suggesting that linked recognition works in both directions, either to establish tolerance to allo, or to break tolerance to self, the critical parameter being the relative number of Treg cells.

Susceptibility of neonatal T cells and adult thymocytes to peripheral tolerance to allogeneic stimuli

We studied the tolerization of neonatal thymocytes (NT), neonatal splenocytes (NS) and adult thymocytes (AT), transferred to syngeneic nude (nu/nu) hosts previously injected with semi‐allogeneic splenocytes, without any supportive immunosuppressive treatment. This protocol allows the study of peripheral tolerance in the absence of the thymus. BALB/c neonatal T cells and ATs were able to expand in syngeneic BALB/c nu/nu mice and functionally reconstituted an allogeneic response, rejecting (BALB/c × B6.Ba) F1 splenocytes transferred 3–4 weeks after injection of BALB/c cells. However, if (BALB/c × B6.Ba) F1 cells were injected into BALB/c nude hosts 30 days before transfer of NT, NS or AT cells, the F1 population was preserved and specific tolerance to B6 allografts was established. Furthermore, transfer to lymphopenic F1 nu/nu showed that tolerance could be established only for neonatal populations, showing that unique properties of neonatal T cells allow their tolerization in both lymphopenic and non‐lymphopenic conditions, in the absence of suppressive immunotherapy. These results bring empirical support to the possibility of T‐cell engraftment in immunodeficient patients showing partial identity with donor major histocompatibility complex (MHC) genes; the manipulation of immunological maturity of donor T cells may be the key for successful reconstitution of immunocompetence without induction of graft‐versus‐host disease.

Modulation of mature B cells in mice following treatment with ouabain.

Ouabain (OUA) is an endogenous hormone released by the adrenal gland under stress situations. Steroid hormones and glucocorticoids have been characterized as selective inhibitors of lymphopoiesis. The present report shows in vivo modulation of mature B cells in bone marrow, spleen and peripheral blood by ouabain. Mice injected intraperitonially (i.p.) with ouabain 0.56 mg/kg for 3 consecutive days displayed, 24 h after last injection, a decreased cellularity in the bone marrow with diminution of the mature B cell subpopulation while the other B cell subpopulations were preserved. Percentually, the myeloid lineage in bone marrow was increased by ouabain. Numbers of mature B lymphocytes in spleen and peripheral blood were reduced following in vivo treatment. In vitro, the B cell populations were not affected. The effects appear to be independent of steroid hormones and strain. The presence of stable levels of glucocorticoids seems to be important because the effects could only be observed from the fourth week animals life, when glucocorticoid levels are stable. These results open new perspectives for a potential use of ouabain as an immunomodulator.

Enhanced renewal of regulatory T cells in relation to CD4+ conventional T lymphocytes in the peripheral compartment

CD4+ Foxp3+ regulatory T (Treg) cells are necessary for the maintenance of self‐tolerance and T‐cell homeostasis. This population is kept at stable frequencies in secondary lymphoid organs for the majority of the lifetime, despite permanent thymic emigration or in the face of thymic involution. Continuous competition is expected to occur between recently thymus‐emigrated and resident Treg cells (either natural or post‐thymically induced). In the present work, we analysed the renewal dynamics of Treg cells compared with CD4+Foxp3‐ conventional T cells (Tconv), using protocols of single or successive T‐cell transfers into syngeneic euthymic or lymphopenic (nu/nu or RAG2−/−) mice, respectively. Our results show a higher turnover for Treg cells in the peripheral compartment, compared with Tconv cells, when B cell‐sufficient euthymic or nude hosts are studied. This increased renewal within the Treg pool, shown by the greater replacement of resident Treg cells by donor counterparts, correlates with augmented rates of proliferation and is not modified following temporary environmental perturbations induced by inflammatory state or microbiota alterations. Notably, the preferential substitution of Treg lymphocytes was not observed in RAG2−/− hosts. We showed that limited B‐cell replenishment in the RAG2−/− hosts decisively contributed to the altered peripheral T‐cell homeostasis. Accordingly, weekly transfers of B cells to RAG2−/− hosts rescued the preferential substitution of Treg lymphocytes. Our study discloses a new aspect of T‐cell homeostasis that depends on the presence of B lymphocytes to regulate the relative incorporation of recently arrived Treg and Tconv cells in the peripheral compartment.

Critical influence of the thymus on peripheral T cell homeostasis.

A tight balance between regulatory CD4+Foxp3+ (Treg) and conventional CD4+Foxp3− (Tconv) T cell subsets in the peripheral compartment, maintained stable throughout most of lifetime, is essential for preserving self‐tolerance along with efficient immune responses. An excess of Treg cells, described for aged individuals, may critically contribute to their reported immunodeficiency. In this work, we investigated if quantitative changes in thymus emigration may alter the Treg/Tconv homeostasis regardless of the aging status of the peripheral compartment.

Extrathymic T cells expand in nude mice following different allogeneic stimuli

We studied extrathymic lymphocyte populations expanded in nude mice after allogeneic stimuli. These were either cells from different tissues or Immunoglobulin (Ig). Although the cells transferred, obtained from Thy-1.1+ donors, were able to induce similar increase in the nude host Thy-1.2+ population, the expanded populations could be qualitatively distinguished from each other by their different expression of mature T cell molecules and by their functional profile. The extrathymic lymphocytes expanded in animals receiving allogeneic fetal thymocytes (FT) were preferentially CD4+ cells and could confer a functional immunocompetent system to the nude host, able to reject allogeneic skin grafts. In contrast, allogeneic adult red blood cells (RBC) led to the expansion of a CD8+ population and to an auto-reactive profile, resulting in the rejection of syngeneic skin grafts by most of the nude hosts. Neither of these profiles was achieved with the other stimuli. These findings support the view that different activation pathways and/or regulatory interactions may lead to the development of distinct extrathymic populations.

Probiotic treatment during neonatal age provides optimal protection against experimental asthma through the modulation of microbiota and T cells

The incidence of allergic diseases, which increased to epidemic proportions in developed countries over the last few decades, has been correlated with altered gut microbiota colonization. Although probiotics may play a critical role in the restoration of gut homeostasis, their efficiency in the control of allergy is controversial. Here, we aimed to investigate the effects of probiotic treatment initiated at neonatal or adult ages on the suppression of experimental ovalbumin (OVA)-induced asthma. Neonatal or adult mice were orally treated with probiotic bacteria and subjected to OVA-induced allergy. Asthma-like symptoms, microbiota composition and frequencies of the total CD4+ T lymphocytes and CD4+Foxp3+ regulatory T (Treg) cells were evaluated in both groups. Probiotic administration to neonates, but not to adults, was necessary and sufficient for the absolute prevention of experimental allergen-induced sensitization. The neonatally acquired tolerance, transferrable to probiotic-untreated adult recipients by splenic cells from tolerant donors, was associated with modulation of gut bacterial composition, augmented levels of cecum butyrate and selective accumulation of Treg cells in the airways. Our findings reveal that a cross-talk between a healthy microbiota and qualitative features inherent to neonatal T cells, especially in the Treg cell subset, might support the beneficial effect of perinatal exposure to probiotic bacteria on the development of long-term tolerance to allergens.

Crucial role for T cell-intrinsic IL-18R-MyD88 signaling in cognate immune response to intracellular parasite infection

MyD88 is the main adaptor molecule for TLR and IL-1R family members. Here, we demonstrated that T-cell intrinsic MyD88 signaling is required for proliferation, protection from apoptosis and expression of activation/memory genes during infection with the intracellular parasite Trypanosoma cruzi, as evidenced by transcriptome and cytometry analyses in mixed bone-marrow (BM) chimeras. The lack of direct IL-18R signaling in T cells, but not of IL-1R, phenocopied the absence of the MyD88 pathway, indicating that IL-18R is a critical MyD88-upstream pathway involved in the establishment of the Th1 response against an in vivo infection, a presently controvert subject. Accordingly, Il18r1−/− mice display lower levels of Th1 cells and are highly susceptible to infection, but can be rescued from mortality by the adoptive transfer of WT CD4+ T cells. Our findings establish the T-cell intrinsic IL-18R/MyD88 pathway as a crucial element for induction of cognate Th1 responses against an important human pathogen.

Enlarged colitogenic T cell population paradoxically supports colitis prevention through the B-lymphocyte-dependent peripheral generation of CD4 + Foxp3 + Treg cells

Intestinal inflammation can be induced by the reconstitution of T/B cell-deficient mice with low numbers of CD4+ T lymphocytes depleted of CD25+Foxp3+ regulatory T cells (Treg). Using RAG-knockout mice as recipients of either splenocytes exclusively depleted of CD25+ cells or FACS-purified CD4+CD25−Foxp3− T cells, we found that the augmentation of potentially colitogenic naïve T cell numbers in the inoculum was unexpectedly beneficial for the suppression of colon disease and maintenance of immune homeostasis. Protection against T cell-mediated colitis correlated with a significant increment in the frequency of peripherally-induced CD4+CD25+Foxp3+ T (pTreg) cells, especially in the mesenteric lymph nodes, an effect that required the presence of B cells and CD4+CD25−Foxp3+ cells in physiological proportions. Our findings support a model whereby the interplay between B lymphocytes and a diversified naïve T cell repertoire is critical for the generation of CD4+CD25+Foxp3+ pTreg cells and colitis suppression.

Comparative analysis of syngeneic and allogeneic mixed lymphocyte reaction of naturally activated and resting T cells

‘Naturally’ activated (NA) or resting T lymphocytes obtained from the spleen of normal BALB/c mice were compared in their capacity to mount a syngeneic mixed lymphocyte reaction (SMLR). Both T‐cell subsets were able to proliferate and secrete IL‐3/GM‐CSF in SMLR cultures. IL‐2 was present in ‘resting’ T‐cell SMLR supernatants, and barely detectable in NA T‐cell SMLR supernatants. Both NA and ‘resting’ T‐cell SMLRs were inhibited with anti‐class II, anti‐CD4, or anti‐IL‐2R MoAbs. NA T cells exhibited a background proliferate and secretory activity in the absence of syngeneic accessory cells. This autonomous activity was susceptible to anti‐CD4, but poorly inhibited with anti‐class II MoAbs. Both NA and ‘resting’ T lymphocytes displayed strong responsiveness to allogeneic stimuli. The analysis of the relative frequency of proliferating cells in the SMLR (BALB/c), or allo‐MLR (B10, B10.A, B10.D2) from NA or ‘resting’ T cells indicated an enrichment for syngeneic reactivity among NA T lymphocytes.