T cell receptor repertoire of CD4+ and CD8+ T cell subsets in the allogeneic bone marrow transplant recipient

Allogeneic bone marrow transplantation (BMT) has become a therapy of choice for the treatment of certain malignancies and hematopoietic disorders. However, immunodeficiencies following BMT continue to cause significant morbidity and mortality. We have compared the T cell receptor (TCR) repertoire of BMT patients and healthy control individuals by staining peripheral blood mononuclear cells with fluorochrome-labeled TCR-specific antibodies. Several patients exhibited a biased pattern of TCR expression atypical of the healthy controls, yet no particular TCR bias characterized all patients. For example, we found that 2%–8% of T cell from healthy individuals expressed the V19 TCR. One BMT patient exhibited V19 expression on more than 60% of peripheral T cells, while additional patients expressed V19 on less than 1% of T cells. The patients with the most extreme skewing of TCR types suffered from graft-versus-host disease. The causes of skewed TCR V expression patterns in BMT patients are not fully understood, yet some researchers have suggested that an oligoclonal expansion of CD8⁺ T cell populations may be largely responsible. To test this hypothesis, we examined the TCR V repertoire of CD4⁺ and CD8⁺ T cell populations. We found that biased V expression characterized both CD4⁺ and CD8⁺ T cell populations, sometimes within a single individual. Thus, therapies directed toward CD8⁺ T cells alone may not fully correct repertoire abnormalities following BMT.     

Monoclonal antibody to a structure expressed on a subpopulation of rat CD8 T cell subsets.

We have developed a monoclonal antibody, RTS-1, which can divide a rat CD8 (+) peripheral T cell population into two functionally distinct subsets. The cell-surface structure defined by this antibody is a glycoprotein with a molecular weight of 220 kDa found to be a high molecular isoform of rat CD45 antigen. CD4 (+) T cells were not stained by RTS-1 antibody. The cytotoxic T cell-enriched population did not express RTS-1 epitope on the cell surface. CD8 (+) spleen cells as well as RTS-1(+)CD8(+)T cells exhibited strong inhibition on mitogen-induced immunoglobulin G production by rat B cells. Furthermore, RTS-1 antibody, but not the control antibody, abolished CD8(+)T cell-mediated inhibition of immunoglobulin G production by rat B cells. These data suggest that RTS-1 antibody recognizes a unique determinant of rat CD45 antigen that is expressed on a fraction of CD8(+) cells.     

Adiponectin promotes human jaw bone marrow mesenchymal stem cell chemotaxis via CXCL1 and CXCL8

Adiponectin (APN) is known to promote the osteogenic differentiation of human jaw bone marrow mesenchymal stem cells (h‐JBMMSCs). However, the underlying mechanism has not been fully elucidated. Previously, we showed that APN could promote h‐JBMMSC osteogenesis via APPL1‐p38 by up‐regulating osteogenesis‐related genes. Here, we aimed to determine whether APN could promote h‐JBMMSC chemotaxis through CXCL1/CXCL8. The CCK‐8, wound healing and transwell assays were used to evaluate the proliferation, migration and chemotaxis of h‐JBMMSCs with or without APN treatment. Chemotaxis‐related genes were screened using RNA‐seq, and the results were validated using real‐time PCR and ELISA. We also performed Western blot using the AMPK inhibitor, WZ4003, and the p38 MAPK inhibitor, SB203580, to identify the signalling pathway involved. We found that APN could promote h‐JBMMSC chemotaxis in the co‐culture transwell system. CXCL1 and CXCL8 were screened and confirmed as the up‐regulated target genes. The APN‐induced CXCL1/8 up‐regulation to promote chemotaxis could be blocked by CXCR2 inhibitor SB225002. Western blot revealed that the phosphorylation of AMPK and p38 MAPK increased in a time‐dependent manner with APN treatment. Additionally, WZ4003 and SB203580 could suppress the APN‐induced overexpression of CXCL1 and CXCL8. The results of the transwell chemotaxis assay also supported the above results. Our data suggest that APN can promote h‐JBMMSC chemotaxis by up‐regulating CXCL1 and CXCL8.     

Dissection and molecular analysis of alloreactive CD8+ T cell responses in allogeneic haematopoietic stem cell transplantation

We applied a cDNA expression screening procedure with cryopreserved non-clonal CD8+ T cell populations (Lennerz et al., Proc. Natl. Acad. Sci. USA 102:16013-8, 2005) to the identification of candidate antigens for graft-versus-host disease (GvHD) and graft-versus-leukaemia (GvL) effects in allogeneic haematopoietic stem cell transplantation (allo-HSCT). In a patient–donor model system with HLA class I disparities, we identified an HLA-B*44 mismatch allele, HLA-B*4405, as the dominant target of alloreactive T cells expanded in vitro from donor peripheral blood mononuclear cells (PBMC). HLA-B*4405-reactive T cells were detectable after multiple in vitro stimulations in the patient’s post-HSCT PBMC. In a patient–donor model with full HLA compatibility, the major target antigen of donor lymphocytes stimulated in vitro with the respective patient’s pre-HSCT PBMC was restricted by HLA-A*0201 and was encoded by TRIM22-442 C, a newly detected polymorphic allele of the tripartite motif family member TRIM22 (synonym: STAF50), preferentially expressed in cells of the haematopoietic system. An arginine(R)-to-cysteine(C) exchange at position 442 generated an immunogenic T cell epitope equivalent to a minor histocompatibility antigen (mHag). TRIM22-442C-specific T cells persisted long-term in the patient’s post-HSCT PBMC. Approximately, 1.3% of Caucasians carry TRIM22.442 C in association with HLA-A*0201. In particular, the knowledge of a large and diverse panel of such mHags may be crucial for further improvement of donor selection and adoptive T cell transfer strategies. The procedure applied herein will help to accelerate and facilitate their identification.     

CD8(+), alphabeta-TCR(+), and gammadelta-TCR(+) cells in the recipient hematopoietic environment mediate resistance to engraftment of allogeneic donor bone marrow

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of alphabeta-TCR(+), gammadelta-TCR(+), alphabeta- plus gammadelta-TCR(+), CD8(+), or CD4(+) cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4(+) T cells. Mice lacking both alphabeta- and gammadelta-TCR(+) cells engrafted without conditioning, suggesting that both alphabeta- and gammadelta-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8(-) cell was targeted by the CyP conditioning. The CD8(+) cell effector function is mechanistically different from that for conventional T cells, and independent of CD4(+) T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.     

Characterization of CD8+ T lymphocytes that persist after peripheral tolerance to a self antigen expressed in the pancreas

As a result of expression of the influenza hemagglutinin (HA) in the pancreatic islets, the repertoire of HA-specific CD8+ T lymphocytes in InsHA transgenic mice (D2 mice expressing the HA transgene under control of the rat insulin promoter) is comprised of cells that are less responsive to cognate Ag than are HA-specific CD8+ T lymphocytes from conventional mice. Previous studies of tolerance induction involving TCR transgenic T lymphocytes suggested that a variety of different mechanisms can reduce avidity for Ag, including altered cell surface expression of molecules involved in Ag recognition and a deficiency in signaling through the TCR complex. To determine which, if any, of these mechanisms pertain to CD8+ T lymphocytes within a conventional repertoire, HA-specific CD8+ T lymphocytes from B10.D2 mice and B10.D2 InsHA transgenic mice were compared with respect to expression of cell surface molecules, TCR gene utilization, binding of tetrameric KdHA complexes, lytic mechanisms, and diabetogenic potential. No evidence was found for reduced expression of TCR or CD8 by InsHA-derived CTL, nor was there evidence for a defect in triggering lytic activity. However, avidity differences between CD8+ clones correlated with their ability to bind KdHA tetramers. These results argue that most of the KdHA-specific T lymphocytes in InsHA mice are not intrinsically different from KdHA-specific T lymphocytes isolated from conventional animals. They simply express TCRs that are less avid in their binding to KdHA.     

CD117+ stem cells play a key role in therapeutic angiogenesis induced by bone marrow cell implantation

Therapeutic angiogenesis can be induced by the implantation of bone marrow mononuclear cells. We investigated the roles of mature mononuclear cell and stem cell fractions in bone marrow in this treatment. Although CD34 is the most popular marker for stem cell selection for inducing therapeutic angiogenesis, we separated CD117-positive cells (CD117+) from mature bone marrow mononuclear cells [CD117-negative cells (CD117-)] from mice using the antibody to the stem cell receptor, because some of the bone marrow stem cells that express CD117+ and CD34- might generate angiogenic cytokines and differentiate into endothelial cells. The angiogenic potency of CD117+ and CD117- cells was investigated in vitro and in vivo. Significantly higher levels of VEGF were secreted from the CD117+ cells than from the CD117- cells (P < 0.001). Most of the CD117- cells died, but the CD117+ cells grew well and differentiated into endothelial cells within 14 days of culture. The CD117+ cells survived and were incorporated in microvessels within 14 days of being implanted into the ischemic hindlimbs of mice, but the CD117- cells did not. The microvessel density and blood perfusion of the ischemic hindlimbs were significantly higher in the CD117+ cell-implanted mice than in the CD117- cell-implanted mice (P < 0.01). The microvessel density in ischemic hindlimbs was also significantly higher in the CD117+ cell-implanted mice than in the total bone marrow cell-implanted mice (P < 0.05). Thus CD117+ stem cells play a key role in the therapeutic angiogenesis induced by bone marrow cell implantation.     

Isolation of primary HIV-1 that target CD8+ T lymphocytes using CD8 as a receptor

HIV-1 use CD4 receptors to infect their primary targets, CD4+ cells, whereas CD8+ cells have a protective role against HIV-1. We recently isolated HIV-1-producing CD8+ clones from two AIDS patients. Here we show that although HIV-1 produced by CD8+ cells maintained the ability to infect CD4+ cells, these viruses were able to infect CD8+ cells independent of CD4. Evidence indicates that these viruses used CD8 as a receptor to infect CD8+ cells. First, expression of CD8 was downmodulated after infection. Second, anti-CD8 antibodies blocked viral entry and replication in CD8+ cells. Finally, resistant cells became susceptible after expression of CD8. Although these viruses used CXCR4 to enter CD4+ cells, it seems that infection of CD8+ cells was independent of CXCR4 or CCR5 co-receptors. Novel changes were observed in envelope sequences of CD8-tropic viruses. These results provide initial evidence that HIV-1 can mutate to infect CD8+ cells using CD8 as a receptor.     

The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8+ T cell differentiation and function

1. Download : Download high-res image (132KB)
2. Download : Download full-size image

     

Regulatory role of CD8+ T lymphocytes in bone marrow eosinophilopoiesis

Background

The aim of the study is to examine the effect of limited and prolonged hyperoxia on neonatal rat lung. This is done by examining the morphologic changes of apoptosis, the expression of ceramide, an important mediator of apoptosis, the expression of inflammatory mediators represented by IL-1β and the expression of 2 proto-oncogenes that appear to modulate apoptosis (Bax and Bcl-2).

Methods

Newborn rats were placed in chambers containing room air or oxygen above 90% for 7 days. The rats were sacrificed at 3, 7 or 14 days and their lungs removed. Sections were fixed, subjected to TUNEL, Hoechst, and E-Cadherin Staining. Sections were also incubated with anti-Bcl-2 and anti-Bax antisera. Bcl-2 and Bax were quantitated by immunohistochemistry. Lipids were extracted, and ceramide measured through a modified diacylglycerol kinase assay. RT-PCR was utilized to assess IL-1β expression.

Results

TUNEL staining showed significant apoptosis in the hyperoxia-exposed lungs at 3 days only. Co-staining of the apoptotic cells with Hoechst, and E-Cadherin indicated that apoptotic cells were mainly epithelial cells. The expression of Bax and ceramide was significantly higher in the hyperoxia-exposed lungs at 3 and 14 days of age, but not at 7 days. Bcl-2 was significantly elevated in the hyperoxia-exposed lungs at 3 and 14 days. IL-1β expression was significantly increased at 14 days.

Conclusion

Exposure of neonatal rat lung to hyperoxia results in early apoptosis documented by TUNEL assay. The early rise in Bax and ceramide appears to overcome the anti-apoptotic activity of Bcl-2. Further exposure did not result in late apoptotic changes. This suggests that apoptotic response to hyperoxia is time sensitive. Prolonged hyperoxia results in acute lung injury and the shifting balance of ceramide, Bax and Bcl-2 may be related to the evolution of the inflammatory process.     

Characterization of a phenotypically distinct subpopulation of Leu-2+ cells that suppresses T cell proliferative responses

Two new monoclonal antibodies (termed 2D2 and D12) have been used to identify and to analyze phenotypically distinct subpopulations of human T cells. The 2D2 antibody recognized an antigenic determinant closely related, if not identical, to that reactive with the anti-Leu-2 monoclonal antibody. The D12 antibody reacted with a variety of cell types, which included a subpopulation of Leu-2+ (2D2+) T cells. These antibodies were used to isolate four phenotypically distinct T cell populations by sequential cell sorter techniques. Functional analyses demonstrated that the 2D2+D12+ subset was unique in its ability to suppress the antigen-induced proliferation of T cells. These cells also suppressed the proliferative responses of other T cell subsets stimulated with mitogens. Pretreatment of 2D2+D12+ T cells with mitomycin C before culture abrogated the suppressor cell activity of these cells. We propose that the cells within the Leu-2+ cytotoxic/suppressor T cell subpopulation that suppress T cell proliferation are phenotypically distinct and express the 2D2+D12+ membrane antigenic phenotype.     

Estimating the size of the dividing stem cell pool after allogeneic bone marrow transplantation

Currently the problem of estimation of the number of pluripotent stem cells reconstituting marrow grafts following bone marrow transplantation, or studies looking at questions of clonal dominance in hematopoietic cell populations, rely on indirect measurement and a simple application of the formula for the sampling variation of a binomial proportion. This approach, from a statistical viewpoint, can be seen to be flawed. It is very easily remedied though and only requires appropriate use of variance stabilizing transformations. These lead to a very simple estimator for the number of hematopoietic stem cells involved in repopulating the marrow and require little in the way of additional calculation. We give the distribution theory for this estimator as well as simple approximations for practical application. As an illustration we rework data recently gathered to address the question as to whether or not reconstitution of marrow grafts in the clinical setting is oligoclonal.     

Somatic cell mapping of T-cell receptor CD3 complex and CD8 genes in cattle

Bovine genes encoding T-cell receptor, CD3, and CD8 molecules have been mapped to syntetic groups using bovine × rodent hybrid somatic cells. T-cell receptor and chains were assigned to bovine syntenic group U5, and the and genes were syntenic with each other and with markers on U13. CD3E and CD3D genes were syntenic with each other and located to bovine syntenic group U19. CD8 was most concordant with markers of syntenic group U16, although the concordancy was only 85% and the assignment must be regarded as tentative. The comparative gene maps of human chromosome 7, bovine syntenic group U13, and mouse chromosomes 6 and 13 suggest extensive evolutionary conservation.     

Virus-specific CD8+ T-cell responses in mice transgenic for a T-cell receptor beta chain selected at random.

The consequences of severely limiting the T-cell receptor (TCR) repertoire available for the response to intranasal infection with an influenza A virus or with Sendai virus have been analyzed by using H-2k mice (TG8.1) transgenic for a TCR beta-chain gene (V beta 8.1D beta 2J beta 2.3C beta 2). Analyzing the prevalence of V beta 8.1+ CD8+ T cells in lymph node cultures from nontransgenic (non-TG) H-2k controls primed with either virus and then stimulated in vitro with the homologous virus or with anti-CD3 epsilon showed that this TCR is not normally selected from the CD8+ T-cell repertoire during these infections. However, the TG8.1 mice cleared both viruses and generated virus-specific effector cytotoxic T lymphocytes (CTL) and memory CTL precursors, though the responses were delayed compared with the non-TG controls. Depletion of the CD4+ T-cell subset had little effect on the course of influenza virus infection but substantially slowed the development of the Sendai virus-specific CTL response and virus elimination in both the TG8.1 and non-TG mice, indicating that CD4+ helpers are promoting the CD8+ T-cell response in the Sendai virus model. Even so, restricting the available T-cell repertoire to lymphocytes expressing a single TCR beta chain still allows sufficient TCR diversity for CD8+ T cells (acting in the presence or absence of the CD4+ subset) to limit infection with an influenza A virus and a parainfluenza type 1 virus.     

Identification of a CD2- CD3+ T cell receptor-gamma+ peripheral blood lymphocyte subpopulation

We have identified, in a healthy individual, a sub-population of human peripheral lymphocytes which surface express a CD3-TCR-gamma complex recognized by anti-Ti gamma A mAb, while being unreactive with a phycoerythrin-conjugated anti-CD2 antibody with T11/1 specificity. Further immunofluorescence analyses performed on uncultured cells indicated that such a putative CD2-CD3+ phenotype was restricted to a fraction of those T lymphocytes which carry a surface receptor of the "second family" (gamma/delta). The actual lack of CD2 expression was confirmed by a subsequent series of cloning experiments which showed that none of the three well characterized CD2 epitopic clusters, namely T11/1, T11/2, and T11/3, were detectable on the surface of the relevant cells. The cultured CD2-, CD3+/TCR gamma + lymphocytes were found to display, as well as their CD2+ counterparts, both non-MHC-restricted cytotoxic function and proliferative responses induced via the gamma receptor complex. In contrast, the proliferative capacity of the CD2-, CD3+/TCR-gamma + cells observed in a culture system designed for in vitro expansion of lymphocytes with undefined specificity was extremely limited. This may relate to an impaired interaction of the CD2- cloned lymphocytes with lymphocyte function-associated (LFA)3+ irradiated cells present in the feeder layer. Further characterization of such minor CD2- T lymphocytes subsets may help to better understand the biologic relevance of the CD2/LFA3 pathway of cell-cell interaction.     

Triggering of effector functions on a CD8+ T cell clone upon the aggregation of an activatory CD94/kp39 heterodimer

Some T lymphocytes express the CD94 Ag, which is known to form heterodimers with members of the NKG2 family. We have studied the expression pattern and function of CD94 heterodimers in different alphabeta or gammadelta T cell clones. Most of the CD94+NKG2A- T cells have a low to intermediate expression of CD94 Ag. The cross-linking of the CD94/NKG2 heterodimer in one of these CD8 alphabeta CD94+NKG2A- T cell clones (K14B06) was able to: 1) increase the intracellular concentration of Ca2+, 2) induce the up-regulation of CD25 Ag expression and the secretion of IFN-gamma, and 3) trigger redirected cytotoxicity in a TCR-independent manner. This activatory property was not shared by any other costimulatory molecule expressed by the K14B06 T cell clone, including CD8, CD28, CD45, CD69, or CD2 Ags. The immunoprecipitation of CD94 heterodimer showed a 39-kDa band with a similar m.w. to the activatory heterodimer found on some NK clones. A novel form of the NKG2 family (NKG2H) was identified in K14B06. NKG2H protein represents an alternative spliced form of the NKG2E gene, displaying a charged residue in the transmembrane portion and a cytoplasmic tail that lacks immunoreceptor tyrosine-based inhibitory motifs. The expression of NKG2H in the cell membrane through its association to CD94 and DAP-12 molecules supports that it could form part of the activatory CD94/Kp39 heterodimer present on K14B06 cells.     

CD4 T cell-mediated alloresistance to fully MHC-mismatched allogeneic bone marrow engraftment is dependent on CD40-CD40 ligand interactions, and lasting T cell tolerance is induced by bone marrow transplantation with initial blockade of this pathway

Costimulatory blockade can be used to promote allogeneic marrow engraftment and tolerance induction, but on its own is not 100% reliable. We sought to determine whether one or the other of the CD4 or CD8 T cell subsets of the recipient was primarily responsible for resistance to allogeneic marrow engraftment in mice receiving costimulatory blockade, and to use this information to develop a more reliable, minimal conditioning regimen for induction of mixed chimerism and transplantation tolerance. We demonstrate that a single anti-CD40 ligand mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance, but does not reliably overcome CD8 CTL-mediated alloresistance. The data suggest that costimulation, which activates alloreactive CTL, is insufficient to activate alloreactive CD4 cells when the CD40 pathway is blocked. The addition of host CD8 T cell depletion to anti-CD40 ligand treatment reliably allows the induction of mixed chimerism and donor-specific skin graft tolerance in 3 Gy-irradiated mice receiving fully MHC-mismatched bone marrow grafts. Thus, despite the existence of multiple costimulatory pathways and pathways of APC activation, our studies demonstrate an absolute dependence on CD40-mediated events for CD4 cell-mediated rejection of allogeneic marrow. Exposure to donor bone marrow allows rapid tolerization of alloreactive CD4 cells when the CD40 pathway is blocked, leading to permanent marrow engraftment and intrathymic tolerization of T cells that develop subsequently.     

Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo

In order to track hematopoetic cells of all lineages unambiguously at all stages of development, we have developed C57BL/6 mice that express a transgene coding for green fluorescent protein (GFP) under control of the human ubiquitin C promoter. These mice, called UBI-GFP/BL6, express GFP in all tissues examined, with high levels of GFP expression observed in hematopoetic cells. UBI-GFP/BL6 mice are unique in that B cells, T cells, and dendritic cells have distinct levels of GFP fluorescence. In cell transfer experiments, leukocytes from UBI-GFP/BL6 mice are readily identified by FACS or fluorescence microscopy. We demonstrate that transplanted UBI-GFP/BL6 dendritic cells are easily identified in secondary lymphoid tissues. Direct interactions between individual dendritic cells and multiple na?ve CD8+ T cells are observed in lymph nodes within 12 h of cell transfer and require loading of the dendritic cells with the appropriate peptide antigen. Dendritic cells undergo specific morphologic changes following interactions with antigen-specific T cells.     

Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses

Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2⁺ cells after intravenous injection and in CD11c⁺ dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8⁺ T-cell response in HLA-A2 transgenic mice and stimulated a CD4⁺ T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A^b. As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.