Takehisa Kaneko

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of theHLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked toHLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of theHLA-D region on a molecular level.

Two distinct subtypes of the HLA-DRw12 haplotypes in the Japanese population detected by nucleotide sequence analysis and oligonucleotide genotyping.

We determined the DNA sequence of the enzymatically amplified second exon of theDRB1 gene of theDRw12 haplotypes derived from three Japanese donors and found two distinct subtypes of theDRw12 haplotype. The two subtypes, designatedDRw12a andDrw12b, had single-base substitutions that predicted one amino acid change at residue number 67. The sequence of theDrw12a andDRw12b subtypes differed from those of the otherDR haplotypes, but in the first hypervariable region of theDRB1 gene the sequences were identical to those of theDRw8(Dw8.1) andDRw8(Dw8.3) haplotypes. TheDRw12a andDRw12b subtypes were detected in a wide range of Japanse donors by genotyping with sequence-specific oligonucleotide probes synthesized according to the DNA sequence of the two subtypes. Results of this study demonstrated that theDRw12 haplotypes in the Japanese population are genetically diverse, as many otherDR haplotypes are.

Human T-cell receptor TCRAV, TCRBV, and TCRAJ sequences newly found in T-cell clones reactive with allogeneic HLA class II antigens

1 Department of Immunology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228, Japan 2 Human Immunogenetics Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98 104, USA 3 ARC Epidemiology Research Unit, University of Manchester, Manchester M13 9PT, England 4 Section for Transplantation Immunology and IIImlunohaematology, Medizinische Universit~itsklinik, W-7400 Ttibingen, Germany 5 Laboratorio di Immunogenetica, Istituto Nazionale per la Ricerca sul Cancro, 16 132 Genova, Italy

Development of immunocompetent lymphocytes in vivo from murine umbilical cord blood cells

Background. Reports concerning the immunological functions of lymphocytes derived from umbilical cord blood cell (UCBC) have been limited. Methods. In murine syngeneic transplantation system using green fluorescent protein transgenic donors, UCBC-derived lymphocytes were studied for their immunological competence. Results. Hematopoietic stem cells (HSC) among UCBC differentiated in the recipients into phenotypically mature T and B lymphocytes. The T lymphocytes were capable of specific recognition of major histocompatibility complex/peptide complex and of the subsequent activation. The antigen-induced CD4+ T cells produced lymphokine upon in vitro antigen stimulation. CD8+ T cells simulated in the mixed lymphocyte culture could lyze specific target cells. Furthermore, RAG2(−/−) mice reconstituted with UCBC mounted specific antibody responses to T-dependent antigen comparable to those by bone marrow chimeras and also rejected allogeneic skin grafts. Conclusion. Collectively the data indicated that T and B lymphocytes derived from UCBC-HSC are fully competent in immunological terms.

Human autoreactive (Th0) CD4+ T-cell clones with cytolytic activity recognizing autologous activated T cells as the target

In attempt to obtain a clue to understanding possible physiological roles played by autoreactive T cells, autoreactive T-cell clones originally derived from an allogeneic mixed lymphocyte culture have been analyzed for their target spectrum, lytic function and cytokine profiles. Five CD4(+) T-cell clones established from allogeneic MLR, in which the stimulator cells shared certain class II MHC antigens with the responder, turned out to be reactive to autologous PBL. Among these, three clones were cytolytic against autologous B-cell line. These three cytolytic autoreactive clones were shown to be capable of specifically lysing autologous activated T cells expressing class II MHC molecules, raising possibility that such autoreactive clones might play a role in negatively regulating T cell responses. Cytolysis by an autoreactive clone 21C5 was inhibited completely by concanamycin A (CMA) known as a specific inhibitor of perforin, suggesting an involvement of the perforin/granzyme system. T-cell clones derived from the same MLC showed distinct correlation between their specificity and lymphokine profiles. Thus, the three cytolytic autoreactive clones belonged to Th0, whereas the two noncytolytic autoreactive clones belonged to Th2 and three alloreactive CD4(+) clones derived from the same culture were of Th1 type.

Allogeneic Recognition of HLA-DRB1 *0406 by T Cells with HLA-DRB 1 *0403: Role of Amino Acid Residue 37 on the β Sheet in T Cell Recognition

Five T cell clones reactive with allogeneic HLA-DR molecules were obtained by stimulating CD4+ T cells (DRB1*0403) with DRB1*0406-homozygous KT13 cells whose DR beta chain differed by a single amino acid residue (37) on the beta sheet from the DRB1*0403 product. Except for one T cell clone which had both auto- and alloreactivities, these clones proliferated by stimulation with KT13 but not with autologous cells, indicating that the single substitution at position 37 on the HLA-DR molecule was sufficient to alter the alloantigenicity of the DR molecule and to elicit an allogeneic T cell response. Two clones reacted with some but not all B cell lines with DRB1*0406, suggesting the possible involvement of a certain peptide whose distribution is restricted to some cells which form the alloantigenic structure recognized by these clones. The two remaining clones showed broad but distinct anti-DR specificity in addition to anti-DRB1*0406 reactivity, suggesting that they recognize the DRB1*0406-peptide complex whose antigenic structures also occur in some combinations of other DRB1 alleles with certain peptides bound to these alleles. The T cell clone with both auto- and alloreactivity was found to react with autologous monocytes but not with autologous B or T cells and to express lower TCR alpha beta than other T cell clones which showed no autoreactivity. The possible recognition molecule for this autoreactive T cell clone is discussed.

DR4 Recognized by PLT Clones

Primed lymphocyte typing (PLT) cells raised in a MLR generally recognize HLA-D region products (1). In this study we examined the serologically defined DR4 by our four PLT clones.