Katsuhiro Tomiyama

Functional alteration of granulocytes, NK cells, and natural killer T cells in centenarians

The immune system in centenarians was characterized as elevated levels in the proportion and number of granulocytes, NK cells, and extrathymic T cells (including NKT cells) in the peripheral blood. Conventional T cells, abundant in youth, were decreased in proportion and number. In addition to this numerical change in centenarians, the function was significantly altered in comparison with that in middle-aged subjects. The phagocytic function and cytokine production of granulocytes in centenarians increased whereas the production of superoxides from granulocytes decreased. This tendency was almost the same in both healthy and unhealthy centenarians. IFN gamma production by NK and extrathymic T cells in centenarians seemed to be augmented and resulted in an elevated level of serum IFN gamma. Possibly due to the effect of this endogenous IFN gamma, the proportion of CD64(+) (Fc gamma RI) cells among granulocytes was elevated. The expansion of CD64 antigens on granulocytes is known to be regulated by IFN gamma and to be associated with their induction of phagocytosis. These results suggest that the immune system of centenarians is not merely impaired, but altered in terms of the number and functions of granulocytes, NK cells, NKT cells.

Enforced Expression of Bcl-2 Restores the Number of NK Cells, But Does Not Rescue the Impaired Development of NKT Cells or Intraepithelial Lymphocytes, in IL-2/IL-15 Receptor β-Chain-Deficient Mice

IL-2/IL-15Rβ-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rβ-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rβ-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rβ-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development.

NK cells contribute to the skin graft rejection promoted by CD4+ T cells activated through the indirect allorecognition pathway.

Rejection of solid organ allografts is promoted by T cells. Recipient T cells can directly recognize intact allo-MHC molecules on donor cells and can also indirectly recognize processed donor-derived allo-peptides presented by recipient antigen-presenting cells in the context of self-MHC molecules. Although CD4(+) T cells primed through the indirect allorecognition pathway alone are sufficient to promote acute allograft rejection, it is unknown how they can mediate graft destruction without cognate recognition of donor cells. In this study, we analyzed the indirect effector mechanism of skin allograft rejection using a mouse model in which SCID recipients bearing MHC class II-deficient skin allografts were adoptively transferred with CD4(+) T cells. Histologically, entire graft necrosis was preceded by mononuclear cell infiltration in the graft epithelia with epithelial cell apoptosis, indicating cell-mediated cytotoxicity against donor cells as an effector mechanism. Beside CD4(+) T cells and macrophages, NK cells infiltrated in the rejecting grafts. Depletion of NK cells as well as blocking of the activating NK receptor NKG2D allowed prolonged survival of the grafts. Expression of NKG2D ligands was up-regulated in the rejecting grafts. These results suggest that NK cells activated through NKG2D contribute to the skin allograft rejection promoted by indirectly primed CD4(+) T cells.

Cytotoxic pathways in the skin allograft rejection by CD4+ T cells.

BACKGROUND Two major mechanisms of T cell-mediated cytotoxicity are known: perforin-dependent and Fas-dependent cytotoxic pathways. Previous studies in vitro demonstrated that CD4+ cytotoxic T lymphocytes use the Fas pathway as a primary cytotoxic mechanism, but the cytotoxic mechanisms used by CD4+ T cells in vivo are unclear. METHODS We examined the cytotoxic pathways of CD4+ T cells in vivo using a skin allograft model, in which athymic nu/nu mice were transplanted with skin allografts and reconstituted with purified CD4+T cells. Fas-deficient and perforin-deficient mice and anti-tumor necrosis factor (TNF)-alpha monoclonal antibody were used for inactivating each cytotoxic pathway in vivo. RESULTS The skin allografts from Fas-deficient mice were readily rejected by the athymic mice reconstituted with purified CD4+ T cells. Perforin-deficient CD4+ T cells could also reject Fas-deficient skin allografts. Furthermore, in vivo treatment with anti-TNF-alpha monoclonal antibody did not prevent the allograft rejection by CD4+ T cells in the absence of both Fas and perforin pathways. CONCLUSIONS These results indicate participation of undefined mechanisms other than Fas, perforin, and TNF-alpha pathways in CD4+ T cell-mediated cytotoxicity in vivo.

Homogeneous epithelial γδ T cell repertoire of the skin is shaped through peripheral selection

In contrast to the T cell receptor (TCR) diversity of major αβ T cells in lymphoid tissues, epithelial T cells of the murine skin, called dendritic epidermal T cells (DETC), express exclusively an invariant γδ TCR. Fetal thymic precursors of DETC immigrate to the skin before birth, and in adult mice T cells expressing the canonical γδ TCR identical to that of DETC are not found in other lymphoid or epithelial tissues. Here, we show that DETC precursors migrate to the gut as well as to the skin during fetal periods, but preferentially survive and expand in the skin after birth. We propose that similar to the thymic selection of the diverse αβ T cell repertoire, ‘peripheral selection’ of the homogeneous epithelial γδ T cell repertoire may be mediated by TCR signaling upon the recognition of the self-ligand, because the ligand for the DETC TCR was expressed only in the skin.

REJECTION OF CULTURED KERATINOCYTE ALLOGRAFTS IN PRESENSITIZED MICE

The fate of cultured keratinocyte (KC) allografts remains controversial. Although prolonged survival of cultured KC allografts in naive mice has been reported, the detailed mechanisms remain undetermined. Furthermore, it was also reported that in the human cultured KC allografts do not survive permanently, and they are rapidly replaced by recipient cells. In the present study, we have addressed this issue and obtained findings that cultured KC allografts survive for a prolonged period in naive mice under the conditions in which reepithelization by recipient cells is prevented. However, the same cultured KC allografts were rejected when they were grafted onto recipients primed with allogeneic spleen cells or full-thickness skin grafts. To clarify the mechanisms behind these findings, the allostimulatory ability of cultured KC and their susceptibility to alloreactive cytotoxic T cells were examined in vitro. In a mixed epidermal cell-lymphocyte reaction, cultured KC were unable to induce allospecific proliferative responses of naive T cells. On the other hand, primed T cells from presensitized mice showed weak but significant proliferative responses against allogeneic KC. It also was confirmed that cultured KC are susceptible to lysis by alloreactive cytotoxic T cells. These data indicate that prolonged survival of cultured KC allografts in naive mice is attributable to a defect in the afferent, but not the efferent, phase of the rejection process that is caused by the weak allostimulatory ability of cultured KC. This assumption was also supported by the finding that spleen cells from the recipient mice bearing long-surviving KC allografts retain in vitro responsiveness against stimulator cells syngeneic to the grafted KC. Taken together, these findings indicate that long-term survival of cultured KC allografts in naive mice may be due solely to the weak allostimulatory ability of cultured KC, but not to loss of susceptibility to alloreactive cytotoxic T cells after culture of KC or induction of tolerance in the recipient mice bearing KC allografts.

Phenotypic and functional modulation of T cells in vivo by extrathymic T cells when T cells with MHC class II disparity were injected into athymic nude mice

TCRhigh cells are generated by the mainstream of T cell differentiation in the thymus, whereas TCRint cells (or NK1.1+ T cells) are generated extrathymically in the liver and by an alternative intrathymic pathway. It is still unknown how these T cell populations interact in vivo with each other. To investigate the interaction of TCRint cells with TCRhigh cells, we used congenitally athymic nude (B6‐nu/nu) mice which carry only TCRint cells in all immune organs. When TCRhigh cells from B6‐C‐H‐2bm12 (bm12) mice (i.e. I‐Abm12) were injected into B6‐nu/nu mice (i.e. 1‐Ab), the expanding T cell population was a mixture of TCRhigh cells of donor origin and TCRint cells of recipient origin. However, 9 Gy‐irradiated nude mice permitted a full expansion of TCRhigh cells which expressed the IL‐2Rα+β+ phenotype, namely, they were at the most activated state. These mice died of acute graft‐versus‐host disease (GVHD) within 5 days. On the other hand, non‐irradiated nude mice suppressed the expansion of TCRhigh cells of donor origin and such TCRhigh cells continued to have the IL‐2Rα±β+ phenotype. These mice could survive but showed signs of chronic GVHD thereafter. In both situations, CD4+αβ T cells expanded irrespective of donor or recipient origin. These results suggest that TCRint cells in the recipient mice possess a regulatory function in relation to donor TCRhigh cells; as a result, fully activated TCRhigh cells acquired the IL‐2Rα+β+ phenotype and injured the host, but TCRhigh cells suppressed in vivo remained as the IL‐2Rα±β+ phenotype and only partially injured the host.

Seeding of dendritic epidermal T cells in the neonatal skin is reduced in 129 strain of mice.

Precursors for Thy-1(+) dendritic epidermal T cells (DETC) develop as Vgamma3(+) T cells in the fetal thymus and become distributed in the adult skin. DETC are variably distributed from site to site and from strain to strain. To elucidate the basis of strain variation, we first compared the density of DETC in the ear epidermis among different mouse strains. In the ear epidermis, we detected the highest level of DETC in C57BL/6 mice, intermediate levels in C3H and CBA/J mice, and the lowest levels in other strains including BALB/c and 129 mice. Although BALB/c and 129+Ter/Sv mice showed higher levels of DETC in the abdomen than in the ear, the levels were significantly lower than C57BL/6 mice. Furthermore, in neonatal abdominal epidermis we detected considerably lower numbers of DETC in BALB/c and 129+Ter/Sv mice than in C57BL/6 mice. In contrast, Vgamma3(+) DETC precursors in the fetal thymus are rather increased in 129+Ter/Sv mice. These results suggest that fewer DETC precursors are seeded in the neonatal skin of BALB/c and 129+Ter/Sv mice and that their expansion in the skin during neonatal to adult stages does not reach the levels in C57BL/6 mice.

Multiple yellowish white papules on the trunk and upper arms

A 32-year-old male was referred to our hospital with a 15-year history of asymptomatic papules which had been gradually increasing in number. There was no history of preceding skin disease in the involved area and no relevant family history. A physical examination revealed multiple nonfollicular yellowish white soft papules with a diameter of 1–5 mm on his trunk and upper arms (Fig. 1). In addition to the papules, the patient exhibited multiple areas of folliculitis on his face and trunk. Radiographic inspection did not find osteopoikilosis. Histological examination of a biopsy specimen obtained from his back showed that the collagen fibers of the upper and middle dermis

Non-paraneoplastic autoimmune subepidermal bullous disease associated with fatal bronchiolitis obliterans

Bronchiolitis obliterans is a small‐airway obstructive lung disease for which immunologically mediated pathogenesis is supposed. Frequent association of bronchiolitis obliterans with paraneoplastic pemphigus is well known, but its association with other autoimmune bullous diseases has not been reported except for a case of anti‐laminin‐332‐type mucous membrane pemphigoid in a patient with chronic graft‐versus‐host disease. We report a case of non‐paraneoplastic autoimmune subepidermal bullous disease associated with fatal bronchiolitis obliterans in a patient without transplantation. Although the patients serum contained immunoglobulin (Ig)A antibodies to the 180‐kDa bullous pemphigoid antigen/type XVII collagen and IgG antibodies to laminin‐332, diagnosis of either linear IgA bullous dermatosis or mucous membrane pemphigoid could not be made because of the failure to detect linear IgA deposition at the basement membrane zone by direct immunofluorescence and the lack of mucous membrane lesions. Physicians should be aware that autoimmune bullous diseases other than paraneoplastic pemphigus can also associate with this rare but potentially fatal lung disease.