Mouse NK1.1⁺αβ TCR⁺ (NKT) cells constitute a unique subset in T-cell population of the thymus and other lymphoid tissues. NKT cells express not only NK-lineage markers but also surface phenotypes of memory/activated T-cells. NKT cells produce a large amount of IL-4 immediately after cross-linking the T cell receptors (TCRs) with anti-CD3 antibody. On the other hand, these cells produce a large amount of IFN-γ upon stimulation through NKR-P1 with anti-NK1.1 antibody. Thus, with their differential productions of cytokines NKT cells may regulate terminal differentiation of T helper cells either to Th1 or to Th2. NKT cells also possess natural cytotoxicity against both autologous and allogeneic immature thymocytes through FasL-Fas interactions.
It has been shown that the NKT cells express invariant Vα14 with a limited Vβ usage and are positively selected by CD1 or TL molecules expressed on CD4⁺8⁺ thymocytes. Thus, the developmental pathway of NKT cells is quite different from that of main-stream T-cells. However, using aly mutant mice we found that radio-resistant thymic component played an important role in generation of NKT cells as obseved in the development of main-stream T-cells.
We also found that a normal population of NKT cells could be generated in the absence of canonical TCRα chain (Vα14Jα281).

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One and a half thousand tiny clusters, filled with one thousand closely packed lymphocytes can be found throughout the murine small and large intestinal mucosa. They are located in crypt lamina propria (cryptopatches; CPs) and can be first detected at 14-17 days after birth. The frequency of DNA replicating cells in CPs is higher than that in Peyer's patches (PPs), is lower than that in the thymic cortex and in almost comparable with that in the thymic medulla. With respect to histogenesis, lymphocyte composition and tissue level of cellular behavior, neither PPs, isolated lymphoid follicles, peripheral LNs, nor thymus are identical with CPs. An outstanding feature of resident CP cells is the conspicuous concentration (up to 70% cells) of c-kit⁺ IL-7R⁺ Thy1⁺ lymphocytes which, in conjunction with the minimal colonization of TCR⁺ and sIgM⁺ lymphocytes, support the notion of lymphoid tissues consisted primarily of precursor T and/or B lymphocytes. CPs are absent in lamina propria of IL-7R-deficient mice, whereas CPs are present in germ-free mice and in athymic (nu/nu), SCID, TCRβ×δ^-/-, RAG-2^-/-, PP-deficient (aly/aly), stem cell factor (Sl/Sl^d) and c-kit (W/W^v) mutant mice. These results indicate that CPs are the first identification of gut-associated murine lymphoid tissues where the generation of IL-7-dependent lymphohematopoietic progenitors for T and/or B cell descendants may start to take place at the age of commencement of weaning.

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In many inflammatory dermatoses, T cells found within the epidermis are regarded as making a major contribution to epidermal damage. However, the presence of T cells within the epidermis during the disease process does not necessarily imply a pathologic role. In mice, T cells residing in the epidermis are thought to play an important role in protecting the epidermis from potentially dangerous immune reactions. To reconcile the apparently discrepant effects of these T cells, we describe the heterogeneity and physiologic functions of epidermal T cells in mice and humans.
In mice congenitally lacking Vγ5⁺ dendritic epidermal T cells (DETC), the epidermis is found to be populated with bone marrow-derived TCR αβ⁺, CD8⁺ DETC. Although it is still unknown whether this subset of DETC could migrate into the epidermis to substitute for the physiologic function of Vγ5⁺ DETC, this subset represents the major fraction of T cells observed in normal human epidermis and those abundantly found in the lesional epidermis of fixed drug eruption (FDE). Localized epidermal injury observed in FDE lesions after clinical challenge with the causative drug would be mediated by activation of these epidermal T cells residing in the FDE lesions. These T cells are shown to kill epidermal cells upon stimulation and utilize a very limited range of TCR Vα and Vβ genes. Epidermal T cells are thus likely to form several T-cell populations with different immunologic functions that are triggered by different modes of stimulation. Immune responses within the epidermis would be regulated by the complex interplay among these diverse epidermal T cells.

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CD4+ T cells have been functionally classified to Th1 and Th2 cells by cytokine production profiles not only in mice but also in humans. Recently, in some respiratory diseases Th1 and Th2 are also classified by several methods, which analyzed cytokine expression patterns on the level of mRNA or secreted protein in BALF. In such analyses it is not clear which cell produces a certain cytokine. Therefore, we have employed a modified Jung's method using flow cytometry which permits analysis of cytokine production at the single cell level. The method is based on the stimulation of T cells by PMA and ionomycin (IM) in the presence of monensin, which inhibits cytokine secretion and results in cytokine accumulation within the cells. After the cells were fixed and stained with anti-cytokine antibodies, intracellular cytokines and cell surface markers of the stimulated cells are simultaneously measured by flow cytometry. We analyzed 3 cases with sarcoidosis and one with chronic eosinophilic pneumonia (CEP) by this method. The percentages of CD4⁺T cells double positive for IFN-γ and IL-2 were 74.9% in sarcoidosis and 15.2% in CEP, and were in between in the normal controls. Our method is suitable to detect cytokine production in individual CD4⁺T cell in normal and disease state, though further investigation should be performed in more cases to understand the immunological state of the chronic respiratory diseases.

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Junctional sequences created by chromosomal translocation in mature B-cell neoplasms, which involve immunoglobulin gene (IG) loci and oncogenes on reciprocal partner chromosomes, are unique to neoplastic cells characterized by particular histological and immunological phenotypes. We present here a novel strategy for detection of the junctional sequences based on long-distance polymerase chain reaction (LD-PCR) amplification. LD-PCR is a general method using primer pairs designed for distinctive regions of the oncogene and IG involved in translocations, and amplifying sequences encompassing the oncogene/IG junction of 2kb up to 30kb in size. LD-PCR is capable of detecting virtually all the important translocations in B-cell neoplasms, including t(8;14)(q24;q32), t(14;18)(q32;q21) and t(3;14)(q27;q32) and its variants. In Burkitt's lymphoma, all materials with the sporadic type c-MYC/IGH fusion showed positive LD-PCR amplification. Restriction analysis and sequencing of the LD-PCR products from BCL2/IGH fusion revealed that breakpoints on BCL2 were distributed over a large region downstream of the BCL2 coding region. 3q27 translocations involving BCL6 and three IGs showed heterogeneous structure at the molecular level. The present ent study suggested that LD-PCR can substitute for time-consuming Southern blot hybridization and cytogenetic analysis in the rapid and sensitive detection of these translocations. Furthermore, as amplified fragments obtained by the LD-PCR contained distinctive regions of oncogenes and IGs, restriction analysis and nucleotide sequencing of the products refined the characteristics of translocations.

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Recently, it has been accepted that Helicobacter pylori (H. pylori) isolated from the gastric mucosa of patients with gastritis in 1983 is highly associated with low-grade gastric lymphoma arising from mucosa-associated lymphoid tissue (L-MALToma). In fact, L-MALToma has been shown to be associated with H. pylori infection in more than 90% of cases. Furthermore, several recent reports demonstrated the regression of L-MALToma after H. pylori eradication with high frequency, although a definite conclusion has not been drawn due to the limited number of cases. Our eradication study also supports this phenomenon. We analyzed the outcome in 20 H. pylori-infected patients with L-MALToma after H. pylori eradication endoscopically, histologically and genetically. Thirteen our of 20 cases (65%) showed changes to chronic gastritis histologically and the mean period until this response was 2.8 months. The endoscopic findings also improved in 12 out of these 13 cases. Althogh 9 cases only showed a monoclonal pattern of IgH and 4 changed to a polyclonal pattern, all of these are responders. These results suggest that H. pylori eradication should be recommended initially for clinical management of L-MALToma, because rapid improvement can be expected. During the follow-up of these patients, however, 3 cases showed the regrowth of lesions and were diagnosed again as L-MALToma histologically. Furthermore, L-MALToma is sometimes invasive and disseminates to regional lymph node, bone marrow and other organs. Therefore, besides the informed consent required from the patients before treatment, the close follow-up using endoscopic examination every 3 months and systemic CT examination every 6 months are needed even if endoscopic biopsy reveals chronic gastritis histologically.

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There are some refractory lymphomas those 5-year overall survival rate is less than 50%, including relapsed or chemotherapy-resistant aggressive NHL, high-intermediate-, or high-risk group of aggressive NHL categorized by International Prognostic Index, mantle cell lymphoma, advanced NK/T cell lymphoma, adult T-cell leukemia/lymphoma (ATL) and advanced and refractory follicular lymphoma. We report here the results of three clinical trials for these refractory lymphomas, as follows; (1) phase I/II trial of cure-oriented high-dose chemoradiotherapy with transplantation of CD34⁺ peripheral blood stem cells purified by the immunomagnetic bead method for refractory hematological malignancies. (2) phase I study of cladribine (2-chlorodeoxyadenosine) in lymphoid malignancies. (3) clinical trial of chimeric anti-CD20 monoclonal antibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma.
(1) Nine patients with relapsed or refractory hematological malignancies including 8 NHL patients were enrolled and treated by high-dose chemoradiotherapy with transplantation of CD34⁺ peripheral blood stem cells purified by the immunomagnetic bead method. In each patient, a median of 1.75×10⁶cells/kg purified CD34⁺ PBSCs (purity; 92%) were infused. Median times to hematopoietic recovery were as same as those in non-purged PBSCT. Most patients experienced only transient toxicities previously observed with the preparative chemotherapy regimens used. Seven patients are alive in remission. This phase I/II trial showed PBSCT with the immunomagnetic bead method to be an active and safe therapy. (2) To clarify the toxicity profiles of cladribine, we conducted a phase I and pharmacological study of cladribine with a schedule of seven-day continuous intravenous infusion every 28 days up to a maximun of three cycles. Anti-tumor effect was observed in the patients with ATL, cutaneous T-cell lymphoma and mantle cell lymphoma. The encouraging results prompted us to plan subsequent phase II studies of cladribine against ATL, hairy cell leukemia and indolent lymphoma. (3) A phase I/II trial of a chimeric mouse human monoclonal antibody to CD20 for CD20 positive recurrent indolent B-cell lymphoma in USA showed a response rate of 50%. A phase II trial has been started for recurrent or relapsed mantle cell lymphoma and indolent B-cell lymphoma in Japan. These three clical trials showed that CD34⁺ PBSCT by the immunomagnetic bead method, cladribine and IDEC-C2B8 are safe and active therapy or drugs, and suggested promising method or agents for refractory lymphomas.

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