Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney-Bone Marrow Transplantation to Induce Transplantation Tolerance.

We examined tolerance mechanisms in patients receiving HLA-mismatched combined kidney-bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high-throughput sequencing of T cell receptor-ß hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non-Tregs revealed marked early enrichment of Tregs (CD3+ CD4+ CD25high CD127low Foxp3+ ) in blood that resulted from peripheral proliferation (Ki67+ ), possibly new thymic emigration (CD31+ ), and, in one tolerant subject, conversion from non-Tregs. Among recovering conventional T cells, central memory CD4+ and CD8+ cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor-reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia-driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.

Mechanisms of donor-specific tolerance in recipients of haploidentical combined bone marrow/kidney transplantation.

We recently reported long-term organ allograft survival without ongoing immunosuppression in four of five patients receiving combined kidney and bone marrow transplantation from haploidentical donors following nonmyeloablative conditioning. In vitro assays up to 18 months revealed donor-specific unresponsiveness. We now demonstrate that T cell recovery is gradual and is characterized by memory-type cell predominance and an increased proportion of CD4⁺ CD25⁺ CD127⁻ FOXP3⁺ Treg during the lymphopenic period. Complete donor-specific unresponsiveness in proliferative and cytotoxic assays, and in limiting dilution analyses of IL-2-producing and cytotoxic cells, developed and persisted for the 3-year follow-up in all patients, and extended to donor renal tubular epithelial cells. Assays in two of four patients were consistent with a role for a suppressive tolerance mechanism at 6 months to 1 year, but later (≥ 18 months) studies on all four patients provided no evidence for a suppressive mechanism. Our studies demonstrate, for the first time, long-term, systemic donor-specific unresponsiveness in patients with HLA-mismatched allograft tolerance. While regulatory cells may play an early role, long-term tolerance appears to be maintained by a deletion or anergy mechanism.

Expression profiles of cytokines and chemokines in murine MDR1a-/- colitis.

OBJECTIVE: MDR1a-/- mice spontaneously develop colitis as the result of imperfect epithelial barrier derived from MDR1a deficiency in the large intestine; however, the pathogenesis is not well understood. This study investigated the expression profiles of cytokines and chemokines in murine MDR1a-/- colitis. METHODS: MDR1a-/- and wild-type FVB mice were monitored from the 6th to the 16th week of age. Production of various cytokines and chemokines in the large intestine and mesenteric lymph node (MLN) cells was examined. RESULTS: Inflammatory cell infiltration, IL-1beta production, and MPO activity were aberrantly enhanced in the tissue of MDR1a-/- mice. Under various stimuli, MLN cells produced higher levels of Th1-type (IFN-gamma, IL-2, and IL-12) and proinflammatory (IL-1beta and TNF-alpha) cytokines. Inflammatory chemokines MIP-2/CXCL2, KC/CXCL1, MIP-1alpha/CCL3, MCP-1/CCL2, and RANTES/CCL5 were also markedly upregulated in the tissue. CONCLUSION: Since the expression profiles of cytokines and chemokines correspond well with those in human IBD, MDR1a-/- mouse is a useful model for the analysis of IBD pathophysiology.

Effect of a novel anti-inflammatory compound, YM976, on antigen-induced eosinophil infiltration into the lungs in rats, mice, and ferrets.

We evaluated the effects of YM976, a selective inhibitor of phosphodiesterase type 4, on antigen-induced eosinophil infiltration into the lungs in rats, mice, and ferrets. In rats, YM976 inhibited the accumulation of eosinophils at an oral ED(50) value of 1.7 mg/kg, and in C57Black/6 mice, exhibited a dose-dependent inhibition at an ED(50) value of 5.8 mg/kg. In the same dose range in the same mouse model, YM976 suppressed interleukin-5 production. We then compared the inhibitory effect of chronic administration with that of single administration in another rat model of eosinophilia induced by repeated antigen exposure. YM976 administered chronically offered more potent inhibition (ED(50) = 0.32 mg/kg p.o.) than a single dose (1.4 mg/kg p.o.). These results indicated that chronic administration is more effective in antigen-induced eosinophilia than a single administration. Emetogenicity is known to be a major adverse effect of phosphodiesterase type 4 inhibitors. We compared the anti-inflammatory activity of YM976 with its emetic activity in ferrets, in which it dose dependently suppressed eosinophil infiltration at an ED(50) value of 1.2 mg/kg, but induced no emesis at 10 mg/kg. This suggested that the compound exhibits a considerable dissociation between its anti-inflammatory and emetic effects. In summary, YM976 inhibited eosinophil infiltration in a dose-dependent manner in rats, mice, and ferrets. In ferrets, it suppressed antigen-induced eosinophil infiltration without emesis. Additionally, we demonstrated that the inhibitory effect on eosinophil infiltration was increased by chronic administration. In conclusion, YM976 is a promising drug for the treatment of diseases involving eosinophil activity, such as asthma.

Antigen dose defines T helper 1 and T helper 2 responses in the lungs of C57BL/6 and BALB/c mice independently of splenic responses.

To investigate the effect of antigen dose on immune response, C57BL/6 and BALB/c mice were sensitized with aluminum hydroxide gel (alum)-precipitated ovalbumin (OVA) then challenged with aerosolized OVA. Low-dose sensitization (less than 8 microg of OVA) elicited T helper 2 (Th2)-type immunoglobulins (Igs) secretion from C57BL/6 mice, including high levels of serum IgE, IgG1 and low levels of IgG2a, while BALB/c mice secreted T helper 1 (Th1)-type Igs, including low levels of IgE, IgG1 and high levels of IgG2a. In contrast, high-dose sensitization (more than 50 microgram) elicited Th1-type Igs secretion in C57BL/6mice, while BALB/c mice exhibited Th2-type Igs secretion. Furthermore, the number of eosinophils infiltrating into the lungs of low-dose OVA-sensitized C57BL/6 mice was significantly greater than in BALB/c mice sensitized with the same amount of OVA. Only a very high dose of OVA (1 mg) could induce greater eosinophil infiltration into the lungs of BALB/c mice compared with C57BL/6 mice. Additionally, low-dose sensitization generated Th2-type cytokines, including high levels of interleukin (IL) -4, IL-5 and a low level of interferon-gamma (IFN-gamma) in the lungs of C57BL/6 mice, while BALB/c mice generated Th1-type cytokines in their lungs, including low levels of IL-4, IL-5 and a high level of IFN-gamma. In contrast, high-dose sensitization elicited Th1-type cytokines production in the lungs of C57BL/6 mice, while BALB/c mice generated Th2-type cytokines in their lungs. Interestingly, splenocyte cultures from C57BL/6 mice produced Th1-type cytokines, while cultures from BALB/c mice produced Th2-type cytokines regardless of OVA sensitization dose (100 ng-1 mg). These results indicate that C57BL/6 and BALB/c mice have different susceptibilities to OVA-sensitization and OVA-induced pulmonary eosinophilia regulated by Th1- and Th2-type cytokines, independent of splenic Th1- and Th2-type cytokines production.

C57BL/6 mice are more susceptible to antigen-induced pulmonary eosinophilia than BALB/c mice, irrespective of systemic T helper 1/T helper 2 responses.

Inflammatory response differences between C57BL/6 and BALB/c mice following ovalbumin (OVA) sensitization and a single challenge were investigated. Serum immunoglobulin (Ig)E and IgG1 levels were higher in C57BL/6 mice than in BALB/c mice. In contrast, IgG2a levels in C57BL/6 mice were lower than in BALB/c mice. Furthermore, the number of eosinophils infiltrating into lungs in C57BL/6 mice was significantly higher than in BALB/c mice after OVA challenge. The levels of the T helper 2 (Th2)-type cytokines interleukin (IL)-4 and IL-5, generated in challenged C57BL/6 lung tissue, were also higher than in BALB/c lung tissue. The participation of IL-4 and IL-5 in the induction of eosinophil infiltration into the lungs was confirmed in both strains of mice by injection of anti-IL-4 and anti-IL-5 monoclonal antibodies (mAbs). However, following OVA stimulation, in vitro IL-4 and IL-5 production in splenocyte cultures from C57BL/6 mice was lower than in splenocyte cultures from BALB/c mice. These results indicate that C57BL/6 mice induce Th2-type responses in the lungs, while BALB/c mice induce T helper 1 (Th1)-type responses in the lungs, despite considerable production of IL-4 and IL-5 from splenocytes. Therefore, local immune responses are more important in the induction of allergic inflammation in the lungs and are different from systemic immune responses, which are thought to depend on genetic background.

Differential susceptibility of C57BL/6 and DBA/2 mice to ovalbumin-induced pulmonary eosinophilia regulated by Th1/Th2-type cytokines.

To test the effect of genotype on immune response, C57BL/6 and DBA/2 mice were sensitized with aluminum hydroxide gel (alum)-precipitated ovalbumin (OVA) and challenged with aerosolized OVA. The serum immunoglobulin (Ig) E and IgG1 levels in C57BL/6 mice were higher than those in DBA/2 mice. In contrast, IgG2a levels in C57BL/6 mice were lower than that in DBA/2 mice. C57BL/6 mice were also much more susceptible than DBA/2 mice to OVA-induced pulmonary eosinophilia. Furthermore, patterns of cytokine generation in lung tissue were different between C57BL/6 and DBA/2 mice after OVA challenge. Th2-type cytokine interleukin (IL-) 4 and IL-5 generation in C57BL/6 mice was higher than that in DBA/2 mice, while Thl-type cytokine interferon-gamma (IFN-gamma) generation in C57BL/6 mice was lower than that in DBA/2 mice. Similar patterns of IL-4 and IL-5, and IFN-gamma production in splenocytes from both strains after OVA stimulation in vitro were also observed. The participation of IL-4 and IL-5, and IFN-gamma in the regulation of eosinophil infiltration into the lung was confirmed by injection of anti-IL-5, -IL-4 and -IFN-gamma monoclonal antibodies. These results indicate that C57BL/6 mice preferentially induce IL-4 and IL-5-mediated Th2-type response, while DBA/2 mice induce IFN-gamma-mediated Thl-type response. Thus, the genotype of laboratory strains partially determines whether Th1- or Th2-type immune responses are elicited.

Costimulatory effect of IL-12 on the activation of naive, memory CD4+ T cells, and Th1 clone.

In the present experiments, costimulatory effects of interleukin 12 (IL-12) and B7-2 on interleukin 2 receptor alpha chain (IL-2R alpha) expression and IL-2-dependent proliferation of naive and memory CD4+ T cells were studied in comparison with the effect of these molecules on Th1 clones. The naive and memory T cells were negatively sorted on the basis of the expression of CD44 and CD45RB, respectively, to diminish the effect of these antibodies on the activation of these cells. When these three T cell populations were stimulated with allogeneic B cells, the addition of IL-12 elicited IL-2R alpha expression and proliferation of all these T cell populations. With anti-B7-2 being included in culture, these responses of naive T cells were abrogated, whereas those of memory T cells and Th1 clones were affected only marginally. When they were stimulated with immobilized anti-CD3, the inclusion of either IL-12 or Chinese hamster ovary cells expressing B7-2 (B7-2CHO) induced IL-2R alpha expression and enhanced IL-2-dependent proliferation of memory T cells, whereas these responses of naive T cells were induced by the inclusion of B7-2CHO and enhanced by the addition of IL-12, although the addition of IL-12 alone did not affect the responses. The responses of Th1 clones were markedly enhanced by IL-12, but not by B7-2CHO, and the enhancement of the proliferation was augmented further by the addition of both IL-12 and B7-2CHO. In IFN-gamma production B7-2 costimulation was required for the enhancing effect of IL-12 on the responses of naive T cells. IL-2 production of all T cell populations was not affected by IL-12 even in the presence of B7-2CHO. These results indicate that IL-12 provides the costimulation for IL-2R alpha expression and IL-2-dependent proliferation of memory T cells and Th1 clones stimulated with TCR ligation, whereas naive T cells require B7-2 costimulation for their response to IL-12. Possible mechanisms of B7-2 costimulation in the response of T cells to IL-12 is discussed.

Early activation signal transduction pathways of Th1 and Th2 cell clones stimulated with anti-CD3. Roles of protein tyrosine kinases in the signal for IL-2 and IL-4 production.

In the present experiments, TCR-CD3-associated early activation signal transduction pathways were examined in Th1 and Th2 clones by the stimulation with soluble monovalent anti-CD3 which resulted in efficient production of IL-2 and IL-4 in Th1 and Th2 cells, respectively. Although protein tyrosine kinases such as Fyn and ZAP-70 were activated in Th1 clones shortly after stimulation, these kinases in Th2 clones were not activated; but, their activity in resting conditions was shown to be decreased by the stimulation. In accordance with these findings, neither phospholipase C-gamma 1 activation nor phosphatidyl inositol-4,5-bisphosphate breakdown was induced in Th2 clones, in contrast to positive responses in Th1 clones. The oscillation of intracellular free Ca2+ concentration ([Ca2+]i) was a common signal for the activation of both Th1 and Th2 clones; however, the [Ca2+]i elevation in Th1 clones was herbimycin A sensitive, whereas that in Th2 was clone resistant, suggesting that the mechanism of the [Ca2+]i elevation in Th2 cells is different from that in Th1 cells in terms of the participation of protein tyrosine kinases. The anti-CD3 stimulation did not cause Lck activation in either the Th1 or Th2 clone, although remarkable activation was induced in both clones following anti-CD4 stimulation, indicating that Lck activation was not required for either IL-2 or IL-4 production of Th cells. Taken together, these results indicate that Th1 and Th2 cells are different from each other in early activation signal transduction pathways, especially in the role of protein tyrosine kinases.

Mechanism of enhanced antigen presentation by B cells activated with anti-mu plus interferon-gamma: role of B7-2 in the activation of naive and memory CD4+ T cells.

B cells activated with anti-mu antibody plus interferon (IFN)-gamma exerted strong antigen presentation activity for T cell proliferation. The enhanced antigen presentation function was shown to be due to the increase in B7-2 expression. When B cells were stimulated with anti-mu, expression of MHC major histocompatibility complex class II, heat-stable antigen (HSA), ICAM-1 and B7-2 was increased. The presence of IFN-gamma further augmented the expression of B7-2 on anti-mu-stimulated B cells. B7-1 was not expressed on B cells under these conditions. The participation of B7-2 in the elicitation of the proliferative response of T cells was confirmed by the inclusion of anti-B7-2 antibody in cultures. The enhanced expression of either HSA or ICAM-1 was shown not to play a major role in the increased B cell antigen presentation capacity. The major T cell population responding to this activated B cell antigen presentation was shown to be CD44low naive CD4+ T cells, whereas CD45RBlow memory CD4+ T cells responded only weakly. The difference in proliferative responses between naive and memory CD4+ T cells was explained by the different efficiency in IL-2 production of these cell populations in response to antigen presentation by B cells activated by anti-mu plus IFN-gamma. These results suggest that IFN-gamma plays an important role in recruitment of naive T cells for an immune response.