Ke-Zheng Dai

The T cell regulator gene SH2D2A contributes to the genetic susceptibility of multiple sclerosis

The T cell specific adapter protein (TSAd) encoded by the SH2D2A gene is involved in the control of T cell activation. The gene is located in the 1q21 region, which has been implicated in susceptibility to experimental allergic encephalomyelitis in the mouse. We therefore evaluated whether a polymorphic GA repeat (GA13–GA33) within the promoter region of the SH2D2A gene shows association to multiple sclerosis (MS). The frequency of the short alleles GA13–16 was increased among 313 Norwegian MS patients compared to 277 healthy controls (0.332 vs 0.249, OR 1.5, Pc = 0.03). Transmission disequilibrium analysis in 146 Scandinavian families with at least two affected sibs showed increased transmission of GA16 to MS patients. No linkage or association of MS to four genetic markers flanking the SH2D2A gene was observed. After activation of naive CD4+ T cells, T cells homozygous for MS associated short alleles displayed lower level of TSAd ex vivo than T cells carrying at least one long allele, which were not associated to MS. Since the SH2D2A protein modulates T cell activation, this may be a mechanism for how short SH2D2A alleles confer susceptibility to develop MS.

The Novel Inhibitory NKR-P1C Receptor and Ly49s3 Identify Two Complementary, Functionally Distinct NK Cell Subsets in Rats

The proximal region of the NK gene complex encodes the NKR-P1 family of killer cell lectin-like receptors which in mice bind members of the genetically linked C-type lectin-related family, while the distal region encodes Ly49 receptors for polymorphic MHC class I molecules. Although certain members of the NKR-P1 family are expressed by all NK cells, we have identified a novel inhibitory rat NKR-P1 molecule termed NKR-P1C that is selectively expressed by a Ly49-negative NK subset with unique functional characteristics. NKR-P1C+ NK cells efficiently lyse certain tumor target cells, secrete cytokines upon stimulation, and functionally recognize a nonpolymorphic ligand on Con A-activated lymphoblasts. However, they specifically fail to kill MHC-mismatched lymphoblast target cells. The NKR-P1C+ NK cell subset also appears earlier during development and shows a tissue distribution distinct from its complementary Ly49s3+ subset, which expresses a wide range of Ly49 receptors. These data suggest the existence of two major, functionally distinct populations of rat NK cells possessing very different killer cell lectin-like receptor repertoires.

Microbial colonization induces oligoclonal expansions of intraepithelial CD8 T cells in the gut

Two populations of CD8+ IEL generally express restricted, but apparently random and non‐overlapping TCR repertoires. Previous studies in mice suggested that this could be explained by a dual origin of CD8+ IEL, i.e. that CD8αβ+ IEL derive from a few peripheral CD8+ T cell lymphoblasts stimulated by microbial antigens in gut‐associated lymphoid tissue, whereas CD8αα+ IEL descend from an inefficient intestinal maturation pathway. We show here that the gut mucosa, instead, becomes seeded with surprisingly broad and generally non‐overlapping CD8 IEL repertoires and that oligoclonality is induced locally after microbial colonization. In germ‐free (GF) rats, both CD8αβ+ and CD8αα+ IEL displayed surprisingly diverse TCR Vβ repertoires, although β‐chain diversity tended to be somewhat restricted in the CD8αα+ subset. CDR3 length displays in individual Vβ‐Cβ and Vβ‐Jβ combinations generally revealed polyclonal distributions over 6–11 different lengths, similar to CD8+ lymph node T cells, and CDR3β sequencing provided further documentation of repertoire diversity. By contrast, in ex‐GF rats colonized with normal commensal microflora, both CD8αβ+ and CD8αα+ IEL displayed oligoclonal CDR3 length distributions for most of the Vβ genes analyzed. Our data suggest that microbial colonization induces apparently random clonal expansions of CD8αβ+ and CD8αα+ IEL locally in the gut.

Two major groups of rat NKR-P1 receptors can be distinguished based on chromosomal localization, phylogenetic analysis and Clr ligand binding.

A major subset of non‐alloreactive NK cells in PVG strain rats is generally low in Ly49 receptors, but expresses the rat NKR‐P1BPVG receptor (previously termed NKR‐P1C). The NKR‐P1B+ NK subset is inhibited by a non‐polymorphic target cell ligand, which we have shown here to be a C‐type lectin‐related molecule (Clr). Clr11 ligates two divergent NKR‐P1B alleles as judged by an NFAT‐driven reporter assay, and inhibits NK‐cell cytotoxicity of NKR‐P1B+ NK cells. Clr11 also interacts with the prototypic NKR‐P1A receptor and exerts a stimulatory influence on NK lysis. NKR‐P1A and B are encoded by adjacent genes in the proximal part of the NK gene complex and show close sequence homology in their extracellular region. They diverge from another pair, NKR‐P1F and ‐G, which is encoded by a second, distal Nkrp1 gene cluster. NKR‐P1F and ‐G bind an overlapping panel of Clr ligands, but not Clr11. Rat Clr molecules appear to be constitutively expressed by hematopoietic cells; expression in tumor cell lines is more variable. The data show the existence of two phylogenetic groups of NKR‐P1 molecules, which demonstrate conservation of ligand‐binding properties independent of signaling function.

Two Structurally Related Rat Ly49 Receptors with Opposing Functions (Ly49 Stimulatory Receptor 5 and Ly49 Inhibitory Receptor 5) Recognize Nonclassical MHC Class Ib-Encoded Target Ligands

The Ly49 family of lectin-like receptors in rodents includes both stimulatory and inhibitory members. Although NK alloreactivity in mice is regulated primarily by inhibitory Ly49 receptors, in rats activating Ly49 receptors are equally important. Previous studies have suggested that activating rat Ly49 receptors are triggered by polymorphic ligands encoded within the nonclassical class Ib region of the rat MHC, RT1-CE/N/M, while inhibitory Ly49 receptors bind to widely expressed classical class Ia molecules encoded from the RT1-A region. To further investigate rat Ly49-mediated regulation of NK alloreactivity, we report in this study the identification and characterization of two novel paired Ly49 receptors that we have termed Ly49 inhibitory receptor 5 (Ly49i5) and Ly49 stimulatory receptor 5 (Ly49s5). Using a new mAb (mAb Fly5), we showed that Ly49i5 is an inhibitory receptor that recognizes ligands encoded within the class Ib region of the u and l haplotypes, while the structurally related Ly49s5 is an activating receptor that recognizes class Ib ligands of the u haplotype. Ly49s5 is functionally expressed in the high NK-alloresponder PVG strain, but not in the low alloresponder BN strain, in which it is a pseudogene. Ly49s5 is hence not responsible for the striking anti-u NK alloresponse previously described in BN rats (haplotype n), which results from repeated alloimmunizations with u haplotype cells. The present studies support the notion of a complex regulation of rat NK alloreactivity by activating and inhibitory Ly49 members, which may be highly homologous in the extracellular region and bind similar class Ib-encoded target ligands.

Phylogenetic and functional conservation of the NKR-P1F and NKR-P1G receptors in rat and mouse

Two clusters of rat Nkrp1 genes can be distinguished based on phylogenetic relationships and functional characteristics. The proximal (centromeric) cluster encodes the well-studied NKR-P1A and NKR-P1B receptors and the distal cluster, the largely uncharacterized, NKR-P1F and NKR-P1G receptors. The inhibitory NKR-P1G receptor is expressed only by the Ly49s3+ NK cell subset as detected by RT-PCR, while the activating NKR-P1F receptor is detected in both Ly49s3+ and NKR-P1B+ NK cells. The mouse NKR-P1G ortholog is expressed by both NKR-P1D− and NKR-P1D+ NK cells in C57BL/6 mice. The rat and mouse NKR-P1F and NKR-P1G receptors demonstrate a striking, cross-species conservation of specificity for Clr ligands. NKR-P1F and NKR-P1G reporter cells reacted with overlapping panels of tumour cell lines and with cells transiently transfected with rat Clr2, Clr3, Clr4, Clr6 and Clr7 and mouse Clrc, Clrf, Clrg and Clrd/x, but not with Clr11 or Clrb, which serve as ligands for NKR-P1 from the proximal cluster. These data suggest that the conserved NKR-P1F and NKR-P1G receptors function as promiscuous receptors for a rapidly evolving family of Clr ligands in rodent NK cells.

Strain-dependent expression of four structurally related rat Ly49 receptors; correlation with NK gene complex haplotype and NK alloreactivity

Natural killer (NK) cells from certain rat strains promptly kill MHC allogeneic lymphocytes in vivo, a rejection phenomenon termed allogeneic lymphocyte cytotoxicity (ALC). ALC can be reproduced in vitro, and is preferentially mediated by a subset of NK cells expressing the Ly49 stimulatory receptor 3 (Ly49s3) in PVG strain rats. Functional studies have suggested that Ly49s3 triggers NK cell alloreactivity, but its importance relative to other Ly49 receptors has not been investigated. In this study, we have characterized three rat Ly49 receptors with close sequence similarity to Ly49s3 in the extracellular region, i.e., Ly49s4, Ly49 inhibitory receptor 3 (Ly49i3), and Ly49i4. Similar to Ly49s3, Ly49s4 mediated cellular activation while Ly49i4 inhibited NK cytolytic function. Ly49s4, -i3, and -i4 all reacted with a previously described anti-Ly49s3 monoclonal antibody (mAb) (DAR13), but not a novel mAb (STOK6), which was shown to be specific for Ly49s3. Expression of these Ly49 receptors varied markedly between inbred strains, in patterns related to their NK gene complex (NKC) haplotype, and ability to mediate ALC. Three major groups of NKC haplotypes could be discerned by restriction fragment length polymorphism analysis. Ly49s3 was present in strains from one of the groups, which corresponded with the “high” ALC responders. Ly49s3 surface expression was also markedly reduced in the presence of its putative MHC class Ib ligand(s) in MHC congenic strains. These data support the notion that Ly49s3 functions as a triggering MHC receptor both in vitro and in vivo. MHC ligands for the other three Ly49 receptors remain to be determined.

A Second Prophylactic Mhc-mismatched Bone Marrow Transplantation Protects Against Rat Acute Myeloid Leukemia (bnml) Without Lethal Graft-versus-host Disease

Background. We have employed a rat model for human acute myeloid leukemia, a promyelocytic leukemia in the BN rat strain (BNML), to develop new protocols for immunotherapy in combination with allogeneic bone marrow transplantation (alloBMT). The status of mixed chimerism in allotransplanted rats provided an opportunity for immunotherapy using alloreactive donor cells. In addition to T or natural killer (NK) cells, we introduced a second infusion of bone marrow cells as prophylactic donor lymphocyte infusions (DLI) to test whether an effective graft-versus-leukemia (GVL) response could be obtained without clinical graft-versus-host disease (GVHD). Methods. BN rats were sublethally irradiated and transplanted with T-cell depleted bone marrow cells from either fully major histocompatibility complex (MHC)-mismatched (PVG) donor rats or MHC-matched (PVG.1N) as controls. Seven days after transplantation, rats were given 500 leukemic cells to mimic minimal residual disease. Additional cellular therapy was given at day +7. The efficiency of DLI was monitored by chimerism analysis in peripheral blood. Results. Rats receiving infusions of NK cells succumbed to leukemia. T-DLI induced complete donor T-cell chimerism and lethal GVHD. A second alloBMT protected against leukemia. This effect was dependent on an MHC incompatibility between the donor and host and also on the presence of alloreactive T cells in the second bone marrow inoculum, resulting in an increased, mixed donor T-cell chimerism. Conclusion. A second prophylactic transplantation influenced the degree of T-cell chimerism to balance favorably between GVL and GVHD. If applicable to humans, repeated alloBMT may provide a novel approach to leukemia therapy.

Structure function analysis of SH2D2A isoforms expressed in T cells reveals a crucial role for the proline rich region encoded by SH2D2A exon 7

BackgroundThe activation induced T cell specific adapter protein (TSAd), encoded by SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd.ResultsTSAd was found to interact with Lck in human CD4+ T cells ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239–256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239–334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239–334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells.ConclusionThese data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239–334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined.

Transcriptional Activation of the SH2D2A Gene Is Dependent on a Cyclic Adenosine 5′-Monophosphate-Responsive Element in the Proximal SH2D2A Promoter

The SH2D2A gene, encoding the T cell-specific adapter protein (TSAd), is rapidly induced in activated T cells. In this study we investigate the regulation of the SH2D2A gene in Jurkat T cells and in primary T cells. Reporter gene assays demonstrated that the proximal 1-kb SH2D2A promoter was constitutively active in Jurkat TAg T cells and, to a lesser extent, in K562 myeloid cells, Reh B cells, and 293T fibroblast cells. The minimal SH2D2A promoter was located between position −236 and −93 bp from the first coding ATG, and transcriptional activity in primary T cells depended on a cAMP response element (CRE) centered around position −117. Nuclear extracts from Jurkat TAg cells and activated primary T cells contained binding activity to this CRE, as observed in an EMSA. Consistent with this observation, we found that a cAMP analog was a very potent inducer of SH2D2A mRNA expression in primary T cells as measured by real-time RT-PCR. Furthermore, activation of SH2D2A expression by CD3 stimulation required cAMP-dependent protein kinase activity. Thus, transcriptional regulation of the SH2D2A gene in activated T cells is critically dependent on a CRE in the proximal promoter region.