CD8 Beta Research Articles

Cross-reactivity influences changes in human influenza A virus and Epstein Barr virus specific CD8 memory T cell receptor alpha and beta repertoires between young and old.

Older people have difficulty controlling infection with common viruses such as influenza A virus (IAV), RNA virus which causes recurrent infections due to a high rate of genetic mutation, and Epstein Barr virus (EBV), DNA virus which persists in B cells for life in the 95% of people that become acutely infected. We questioned whether changes in epitope-specific memory CD8 T cell receptor (TCR) repertoires to these two common viruses could occur with increasing age and contribute to waning immunity. We compared CD8 memory TCR alpha and beta repertoires in two HLA-A2+ EBV- and IAV-immune donors, young (Y) and older (O) donors to three immunodominant epitopes known to be cross-reactive, IAV-M158-66 (IAV-M1), EBV-BMLF1280-288 (EBV-BM), and EBV-BRLF1109-117 (EBV-BR). We, therefore, also designed these studies to examine if TCR cross-reactivity could contribute to changes in repertoire with increasing age. TCR high throughput sequencing showed a significant difference in the pattern of TRBV usage between Y and O. However, there were many more differences in AV and AJ usage, between the age groups suggesting that changes in TCRα usage may play a greater role in evolution of the TCR repertoire emphasizing the importance of studying TRAV repertoires. With increasing age there was a preferential retention of TCR for all three epitopes with features in their complementarity-determining region (CDR3) that increased their ease of generation, and their cross-reactive potential. Young and older donors differed in the patterns of AV/AJ and BV/BJ pairings and usage of dominant CDR3 motifs specific to all three epitopes. Both young and older donors had cross-reactive responses between these 3 epitopes, which were unique and differed from the cognate responses having features that suggested they could interact with either ligand. There was an increased tendency for the classic IAV-M1 specific clone BV19-IRSS-JB2.7/AV27-CAGGGSQGNLIF-AJ42 to appear among the cross-reactive clones, suggesting that the dominance of this clone may relate to its cross-reactivity with EBV. These results suggest that although young and older donors retain classic TCR features for each epitope their repertoires are gradually changing with age, maintaining TCRs that are cross-reactive between these two common human viruses, one with recurrent infections and the other a persistent virus which frequently reactivates.

Expression and polymorphisms of CD8B gene and its associations with body weight and size traits in sheep

The growth traits are economically important traits in sheep. Improving growth rates will increase the profitability of producers. The aim of this study was to identify alleles of CD8B (encoding T-cell surface glycoprotein CD8 beta chain) that are aberrantly expressed in different tissues and to assess the effects and associations of its different genotypes on weight and size traits in sheep. Using quantitative real-time reverse transcription PCR arrays, expression profiling of CD8B was performed in various organs and tissues. CD8B was ubiquitously expressed, with very high expression in the lung, spleen, lymph, duodenum, and liver. One intronic mutation (chr3:62,718,030 (Oar_rambouillet_v1.0, same below) G > A) was identified using pooled DNA sequencing. Subsequently, the variants (AA, AG, and GG) were genotyped using the KASPar® PCR single nucleotide polymorphism (SNP) genotyping system. The results of association analysis with body weight and body size traits in 1304 sheep showed that increases in multiple phenotypic traits correlated with the AA genotype (body weight, p < 0.05; body length, p < 0.05). Thus, SNP chr3:62,718,030 G > A is a promising molecular marker for marker-assisted selection in sheep breeding.

The role of stromal immune microenvironment in the progression of ductal carcinoma in situ (DCIS) to invasive breast cancer

AimThe first aim of the study was to compare the scores and types of stromal immune cells in 30 patients with primary DCIS and in the same patients after invasive breast recurrence in order to assess possible differences in both during tumor progression. The second aim was to evaluate possible differences in stromal cells of 30 patients with primary DCIS before progression and in the control group of 11 DCIS patients without recurrence during long-term follow-up.Material and methodsEvaluation of tumor-infiltrating lymphocytes (TILs) and immunohistochemical stains for immune cell markers CD4, CD8, CD20, CD138, FOXP3, CD163 and TGF beta was performed on the stroma of primary DCIS before progression, invasive breast cancer of the same patients after progression and DCIS without progression.ResultsThe comparison of stromal cells in 30 patients with initial DCIS and its invasive recurrence revealed an increased level of CD20 + immune cells (median score 5% vs. 17%, respectively, p < 0.001) and CD163 + cells (median score 1% vs. 5%, respectively, p < 0.001) in invasive breast cancer. The comparison of stromal cells in 30 patients with initial DCIS before recurrence and the control group of 11 patients with DCIS without recurrence showed statistically significant difference for CD138 + cells, which were more prevalent in patients with worse prognosis (median score 0 vs. 2%, respectively, p < 0.001). No similar relationship was found for the other tested cells as well as for TGF-beta.ConclusionsCD138 + immune cells that were more prevalent in patients with a worse prognosis should be explored in further studies to confirm or exclude their role as a potential biological marker of DCIS invasive recurrence.

Fine tuning CD8+ T cell functional avidity by the PD-1/SHP2 axis

Abstract T cells of high functional avidity recognize low antigen levels and have the most effective cytotoxic responses against viruses and tumor cells. Understanding the molecular regulations of T cell functional avidity is essential to aid novel vaccine design as well as adoptive immunotherapy by improving the T cell quality. Programmed cell death protein 1 (PD-1) is known to negatively regulate the signal strength of T cell receptor (TCR). Activated PD-1 recruits Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (Shp2), which dephosphorylates kinases involved in TCR signaling. However, it is unclear whether PD-1/SHP2 can modulate T cell functional avidity. To address this question, we treated splenocytes from OT-1 transgenic mice with anti-PD-1 or SHP099 (SHP2 inhibitor), or a combination of both for 3 days in a selective culture condition of APC-pulsed stimulation with 10−6 M or 10−9 M SINNFEKL, respectively. Our preliminary data showed that blocking SHP2/PD-1 enhanced functional avidity and antigen sensitivity of CD8+ T cell in vitro when stimulated with limited SINNFEKL antigen. Blocking of PD1/SHP2 also enhanced CD8 beta expression, which has been shown correlated with high functional avidity CD8+ T cells. These data suggest that PD1/SHP2 is involved in regulating T cell functional avidity.

High TCR Vbeta Skewing in CD4+ and Low in CD8+ Is Associated with Development of Chronic Graft-Versus-Host Disease

IntroductionChronic graft-versus-host disease (cGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Perturbations in immune reconstitution following allo-HSCT are believed to be integral to the pathophysiology of cGVHD. We hypothesized that skewing in T cell repertoire around day 80 would be predictive of cGVHD, while maintenance of a broad repertoire would be protective. One method used to assess skewing is the Gini Index, a conventional measure of income inequality. In this prospective study, we utilized a rapid FACS based method of TCR Vβ repertoire analysis within CD4+ and CD8+ in patients at day 80-110 post allo-HSCT to determine whether preferential TCR skewing predicts the development of cGVHD.MethodsPeripheral blood samples were obtained from 79 consented patients at day 80-110 following allo-HSCT at The University of Texas MD Anderson Cancer Center from October 2012 to November 2015. All patients were in remission of their disease at the time of blood sampling. Fresh PBMC were analyzed by FACS after staining for CD3, CD4, CD8 and IOTest Beta Mark 24 TCR Vβ kit. The TCR Gini Index scores (GS) were calculated for the T cell subsets, and ranged from 0 to 100. Low GS indicate more equal distribution of TCR-Vβ families (i.e. broad repertoire), whereas high GS reflect unequal distribution of TCR-Vβ families (i.e. repertoire skewing). Predictive thresholds of GS were identified in quartiles analysis and confirmed by Receiver Operating Characteristic (ROC) curve.The association between GS and the rate of cGVHD since the date of blood sampling was evaluated using competing-risks regression analysis. The diagnosis of cGVHD was based on the 2014 National Institutes of Health guidelines.ResultsThe median age at transplant was 53 years (range 21-75) and 60% of patients were male. The most common transplant indication was AML/MDS (52%) followed by lymphoma (16%), ALL (14%), and other (18%). Graft source was peripheral blood (54%), bone marrow (43%) and umbilical cord blood (UCB) (3%) with donors being matched unrelated (65%), matched-related (24%), mismatch related (9%) and UCB (3%). Forty three (54%) patients were not in remission at the time of transplant. At the time of enrollment, 47% had history of grade II-IV acute GVHD and 44% were on systemic steroids. At a median follow up of 2 years since blood sampling, 16 patients developed cGVHD (cumulative incidence (CI) 23%; 95% Confidence Interval: 15-35). The median time from HSCT to cGVHD was 174 days (range 111-551 days). None of the clinical or demographic characteristics listed above, except history of acute GVHD, was significantly associated with the incidence of cGVHD. GS were significantly predictive of the incidence of cGVHD. The 2 year %CI of cGVHD was 47% in patients who had CD4+ TCR GS >55% (n=19) compared with 16% in those with CD4+ GS ≤55% (n=60) (hazard ratio (HR) =3.4, P=0.01). In contrast to CD4+, lower CD8+ GS predicted for higher incidence of cGVHD. The 2 year %CI of cGVHD was 39% in patients (n=34) with CD8+ GS ≤60% compared with 10% in patients (n=45) with CD8+ GS >60% (HR=4.5, P=0.009). When both CD4+ and CD8+ GS were considered in combination, the 2 year incidence of cGVHD was highest in patients (n=6) with both low CD8+ (≤60%) and high CD4+ (>55%) GS, and lowest in patients (n=32) with high CD8+ (>60%) and low CD4+ (≤55%) GS (%CI: 83% vs 4%, P=0.001). The combined effect of high CD4+ and low CD8+ TCR skewing was more pronounced in patients with a history of grade II-IV acute GVHD. Low CD8+ (≤60%, n=28) or high CD4+ (>55%, n=13) GS occurring separately were associated with comparable rates of cGVHD (%CI: 31% and 27%, p=0.8), irrespective of history of grade II-IV acute GVHD (Figure).ConclusionsHigh CD4+ and low CD8+ TCR Gini indices were independent predictors of cGVHD. Our results indicated that the higher skewing of CD8+ TCR appeared to be protective of cGVHD and the opposite for CD4+. Our study is the first to test the usefulness of FACS-based TCR measurement and Gini Index score in a clinical setting to help predict the development of cGVHD and provide greater insights into the pathogenesis of cGVHD. Using this convenient FACS-based method to identify patients who are at risk of developing cGVHD could be beneficial from development of effective preventive or therapeutic strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Structural analysis of fish versus mammalian hemoglobins: Effect of the heme pocket environment on autooxidation and hemin loss

The underlying stereochemical mechanisms for the dramatic differences in autooxidation and hemin loss rates of fish versus mammalian hemoglobins (Hb) have been examined by determining the crystal structures of perch, trout IV, and bovine Hb at high and low pH. The fish Hbs autooxidize and release hemin approximately 50- to 100-fold more rapidly than bovine Hb. Five specific amino acid replacements in the CD corner and along the E helix appear to cause the increased susceptibility of fish Hbs to oxidative degradation compared with mammalian Hbs. Ile is present at the E11 helical position in most fish Hb chains whereas a smaller Val residue is present in all mammalian alpha and beta chains. The larger IleE11 side chain sterically hinders bound O(2) and facilitates dissociation of the neutral superoxide radical, enhancing autooxidation. Lys(E10) is found in most mammalian Hb and forms favorable electrostatic and hydrogen bonding interactions with the heme-7-propionate. In contrast, Thr(E10) is present in most fish Hbs and is too short to stabilize bound heme, and causes increased rates of hemin dissociation. Especially high rates of hemin loss in perch Hb are also due to a lack of electrostatic interaction between His(CE3) and the heme-6 propionate in alpha subunits whereas this interaction does occur in trout IV and bovine Hb. There is also a larger gap for solvent entry into the heme crevice near beta CD3 in the perch Hb (approximately 8 A) compared with trout IV Hb (approximately 6 A) which in turn is significantly higher than that in bovine Hb (approximately 4 A) at low pH. The amino acids at CD4 and E14 differ between bovine and the fish Hbs and have the potential to modulate oxidative degradation by altering the orientation of the distal histidine and the stability of the E-helix. Generally rapid rates of lipid oxidation in fish muscle can be partly attributed to the fact that fish Hbs are highly susceptible to oxidative degradation.

Changes in thymus size, cellularity and relation between thymocyte subpopulations in young adult rats induced by Somatostatin-14

The role of somatostatin on inhibition of both normal and tumor cell cycle, secretion of endocrine and exocrine cells, as well as induction apoptosis is well documented. However, its effect on T cell development and thymic structure is not fully clarified. In order to investigate the influence of somatostatin in vivo on the thymus structure and T cell development, the young adult Albino Oxford male rats were intracerebroventriculary treated with somatostatin-14. We examined the thymus compartments and its cellularity, through assessment of morphometric parameters by stereological method, and the relation between thymocytes subpopulations, over expression of CD4, CD8 and T-cell receptor (TCR) alpha beta by flow cytometry. Additionally, we also determined the body and thymus weight of the rats, during the first three months of life, to define the time of SRIH-14 application. A decrease of relative thymus weight from the fourth weeks of postnatal life, and an unchanged relative thymus weight obtained in treated group indicates that SRIH-14 in young adult rats inhibits growth of whole organism, not only thymus. The changes in the absolute number and numerical density of cortical thymocytes indicate that SRIH-14 alters the true lymphoid tissue. SRIH-14 changes relation between thymocyte subsets, increase number of CD4(-)CD8(-)TCR alpha beta(-) and CD4(-)CD8(+)TCR alpha beta(hi) thymocyte subsets as well as the CD4(-)CD8(-)TCR alpha beta(low/hi) thymocytes, while decrease number of CD4(+)CD8(+) TCR alpha beta(-/low/hi) thymocyte subsets. These results indicate that somatostatin-14 is not involved in the control of the physiologic involution of the thymus, although induces thymic weight loss through the reduction of true lymphoid tissue. In addition, changes in frequency of thymocyte subpopulations, especially immature cells, indicate that SRIH-14 modulates thymocytes development and maturation.

T-LYMPHOCYTE PROFILES IN FIV-INFECTED WILD LIONS AND PUMAS REVEAL CD4 DEPLETION

Feline immunodeficiency virus (FIV) is a lentivirus related to human immunodeficiency virus (HIV) that causes feline AIDS in the domestic cat (Felis catus). Serological surveys indicate that at least 25 other species of cat possess antibodies that cross-react with domestic cat FIV. Most infected nondomestic cat species are without major symptoms of disease. Long-term studies of FIV genome variation and pathogenesis reveal patterns consistent with coadaptation of virus and host in free-ranging FIV-Ple-infected African lions (Panthera leo) and FIV-Pco-infected pumas (Puma concolor) populations. This report examined correlates of immunodeficiency in wild and captive lions and pumas by quantifying CD5(+), CD4(+), and CD8(+) T-cell subsets. Free-ranging FIV-Ple-infected lions had immunofluorescence flow cytometry (IFC) profiles marked by a dramatic decline in CD4(+) subsets, a reduction of the CD4(+)/CD8(+) ratio, reduction of CD8(+)beta(high) cells, and expansion of the CD8(+)beta(low) subset relative to uninfected lions. An overall significant depletion in CD5(+) T-cells in seropositive lions was linked with a compensatory increase in total CD5(-) lymphocytes. The IFC profiles were altered significantly in 50% of the seropositive individuals examined. The FIV-Pco-infected pumas had a more generalized response of lymphopenia expressed as a significant decline in total lymphocytes, CD5(+) T-cells, and CD5(-) lymphocytes as well as a significant reduction in CD4(+) T-cells. Like lions, seropositive pumas had a significant decline in CD8(+)beta(high) cells but differed by not having compensatory expansion of CD8(+)beta(low) cells relative to controls. Results from FIV-infected lions and pumas parallel human and Asian monkey CD4(+) diminution in HIV and SIV infection, respectively, and suggest there may be unrecognized immunological consequences of FIV infection in these two species of large cats.

Characterization of the varicella zoster virus (VZV)-specific intra-ocular T-cell response in patients with VZV-induced uveitis

Varicella zoster virus (VZV) is a well-known cause of infectious uveitis. The aim of this study was to characterize the VZV-specific T-cell response in eyes of patients with VZV-induced uveitis. T-cell lines (TCL) were generated by mitogenic stimulation of intra-ocular fluid (IOF) samples obtained from eight patients with VZV-induced uveitis. Two patients with herpes simplex virus (HSV)-induced uveitis were included as disease controls. Characterization of individual T-cells in the TCL was performed by stimulating the TCL with mock, HSV-1 and VZV antigen pulsed autologous B cells and subsequent flow cytometric analyses. Virus specificity and phenotype of the T-cells were identified by simultaneous detection of intracellular gamma interferon and cell surface markers CD4, CD8, CD3 or T-cell receptor (TCR) beta chain variable region (TCRBV) usage. The TCL obtained from patients with HSV-1-induced uveitis contained higher numbers of T-cells reactive to HSV-1 compared to VZV. VZV-specific T-cells were detected in all TCL of the patients diagnosed with VZV-induced uveitis. Four out of six TCL obtained from patients with VZV-induced uveitis that were assayed for both viruses, contained higher numbers of T-cells reactive to VZV compared to HSV-1. Detailed analyses of the TCL of two patients demonstrated that the VZV reactivity within the assayed TCL was dominated by T-cells expressing one specific TCRBV gene. The data implicate that VZV-reactive T-cells infiltrate and participate in the local inflammatory response in eyes of patients with VZV-induced uveitis.

Self-reactivity in thymic double-positive cells commits cells to a CD8 alpha alpha lineage with characteristics of innate immune cells.

Thymocytes displaying self-reactive T cell receptors usually undergo negative selection in the thymus. Here we demonstrate that agonist peptides can promote positive selection of immature double-positive thymocytes into distinct lineages, varying with the agonist concentration and the animal's age. Microarray gene expression analyses showed broad transcriptional alterations in a set of transcripts associated with the innate immune system, as well as silencing of CD8 beta expression. The resulting CD8 alpha alpha T cells showed a rapid effector cytokine response. Hence, T cells displaying self-reactive receptors can have the gene expression profile and phenotypic characteristics of innate immune cells.

Patterns of gene expression in the developing chick thymus.

Gene expression in thymic T cells during late embryogenesis and early growth in chicks was examined using cDNA microarrays. Gene expression patterns were profiled into nine clusters by using self-organizing maps (SOM) clustering analysis. The expression patterns for a set of genes confirmed current information on the development of immune response. Expression of cell surface markers (MHC class I alpha chain, MHC class II associated invariant chain, CD8 beta chain, CD18, and beta2-microglobulin), and genes involved in the innate immune response (NK lysin-like) increased with age, and these patterns were consistent with an increase in the immune responsiveness of the young chick. The expression of cytokine receptor common gamma chain (gammac), death receptor-3 (DR3), and TCR alpha chain increased up to 1 day of age and then decreased. DR3 could play a role in the apoptosis during T-cell maturation, while the differential expression of TCR genes could reflect regulation of the rearrangement of TCR genes and TCR-mediated signal transduction during T cell development. Three genes coding for previously uncharacterized proteins are included in the clusters. These gene expression profiling studies provide background information on the developing chick immune system and provide preliminary functional information on unknown proteins.

CD3δ Establishes a Functional Link between the T Cell Receptor and CD8

T cells expressing T cell receptor (TCR) complexes that lack CD3 delta, either due to deletion of the CD3 delta gene, or by replacement of the connecting peptide of the TCR alpha chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Because the same defects have been observed in mice expressing no CD8 beta or tailless CD8 beta, we examined whether CD3 delta serves to couple TCR.CD3 with CD8. To this end we used T cell hybridomas and transgenic mice expressing the T1 TCR, which recognizes a photoreactive derivative of the PbCS 252-260 peptide in the context of H-2K(d). We report that, in thymocytes and hybridomas expressing the T1 TCR.CD3 complex, CD8 alpha beta associates with the TCR. This association was not observed on T1 hybridomas expressing only CD8 alpha alpha or a CD3 delta(-) variant of the T1 TCR. CD3 delta was selectively co-immunoprecipitated with anti-CD8 antibodies, indicating an avid association of CD8 with CD3 delta. Because CD8 alpha beta is a raft constituent, due to this association a fraction of TCR.CD3 is raft-associated. Cross-linking of these TCR-CD8 adducts results in extensive TCR aggregate formation and intracellular calcium mobilization. Thus, CD3 delta couples TCR.CD3 with raft-associated CD8, which is required for effective activation and positive selection of CD8(+) T cells.

In vitro production and characterization of partly assembled human CD3 complexes.

Pairwise assembly of human CD3 chains takes place in the endoplasmic reticulum of T cells. Subsequently, the CD3 heterodimers form complexes with Ti alpha and Tiss chains forming hexameric Ti alpha beta CD3 gamma epsilon delta epsilon complexes. Finally, association with the zeta 2 homodimer occurs in Golgi apparatus before the fully assembled T-cell receptor is transported to the cell surface. To study the structural properties of the human CD3 chains, we have developed new methods to produce and fold the extracellular domains of CD3 gamma, CD3 delta and CD3 epsilon. Proteins were expressed in Escherichia coli as denatured chains and de novo folded in vitro. CD3 gamma and CD3 epsilon folded as soluble monomers, whereas CD3 delta did not yield any soluble proteins. When folding the chains pairwise, soluble CD3 gamma epsilon and CD3 delta epsilon heterodimers could be isolated, whereas CD3 gamma delta heterodimers were not produced. Using antibodies as structural probes, we identified two different types of antigenic epitopes that were dependent on heterodimerization. Our data indicate that CD3 epsilon undergoes a conformational change after dimerization with CD3 gamma or CD3 delta. Furthermore, we demonstrated that the CD3 gamma epsilon heterodimer could be purified using immunoaffinity chromatography.

Diversity of MICA (PERB11.1) and HLA haplotypes in Northeastern Thais.

MICA or PERB11.1 is a polymorphic major histocompatibility complex (MHC) class I-related gene located 46 kb centromeric of the HLA-B gene in the HLA class I region. It is expressed mainly in gut epithelial cells, keratinocytes, endothelial cells, fibroblasts and monocytes, and is upregulated by heat stress. MICA has been found to interact with gamma delta T cells, alpha beta CD8(+) and natural killer (NK) cells bearing the NKG2D/DAP10 receptor. The MICA gene displays a high degree of polymorphism with at least 54 alleles. In the present study, polymorphic exons 2, 3 and 4 of the MICA gene were analyzed using sequencing based typing (SBT) in 255 unrelated healthy northeastern Thais. Thirteen previously reported MICA alleles were detected. MICA*008, *010, *002 and *019 were highly predominant with the allele frequencies of 21.4%, 18.2%, 17.6% and 15.3%, respectively. Five of these 13 MICA alleles show significantly different frequencies from those of the Japanese and Caucasian populations. Interestingly, MICA052, which is a very rare allele in other populations, was prevalent with the allele frequency of 8.2%, mainly on the HLA haplotype carrying HLA-B*13 in this population. Strong linkage disequilibria were observed between MICA and HLA-B, as similarly observed in other populations, namely MICA*010-B*4601, MICA052-B*13, MICA*002-B*5801, and MICA*019-B*15 (1502, 1508, 1511, 1515, 1528, 1530). A large variety of three-locus (MICA - HLA-B - HLA-Cw) and six-locus (HLA-DQB1 - HLA-DRB1 - MICA - HLA-B - HLA-Cw - HLA-A) haplotypes were recognized in the northeastern Thai population. This is the first report on MICA allelic distribution in Southeast Asian populations. These data will provide the important basis for future analyses on the potential role of the MICA gene in disease susceptibility and transplantation matching in Southeast Asian populations.

T lymphopaenia in relation to body mass index and TNF‐α in human obesity: adequate weight reduction can be corrective

Although individuals with obesity are susceptible to infection, the underlying causes have not been fully identified. To investigate whether obesity affects immunity, we studied subjects with isolated obesity. Thirty-four obese persons from our outpatient obesity clinic and 50 nonobese healthy control subjects were studied. The effects of weight reduction were evaluated in obese subjects on a very-low-energy diet. We examined blastogenic response, lymphocyte subsets, circulatory TNF-alpha, soluble TNF-alpha receptor 1, soluble TNF-alpha receptor 2, and in vitro TNF-alpha production in obesity. Lymphocyte subsets were analysed with flowcytometry. TNF-alpha and soluble TNF receptors levels were assayed using commercially available enzyme-linked immunosorbent assay kits. Blastogenic responses to phytohemagglutinin or concanavalin A of T cells, CD3(+), CD4(+), CD8(+), CD4(+)CD45RO(+), and TCR alpha beta T cells were significantly diminished in obese subjects. Strong negative correlations were observed between TCR alpha beta and body weight and BMI in obese subjects. Circulatory levels of TNF-alpha, soluble TNF-alpha receptors, and in vitro TNF-alpha production were significantly increased compared to nonobese subjects. In obese subjects, there were significant positive correlations between serum levels of TNF-alpha and waist-hip ratio, serum levels of soluble TNF-alpha receptor 1 and body weight, soluble TNF-alpha receptor 2 and BMI, and soluble TNF-alpha receptor 2 and waist-hip ratio. The T cell responses and previously reduced non-CD8 T cell subsets were increased significantly following weight reduction. Our results suggest that subsets of T cell populations and their function may be reduced in human obesity, and that this may be related, at least in part, to the elevated TNF-alpha production. Furthermore, this T cell dysfunction can be recovered by adequate weight reduction.

Human CD8 beta, but not mouse CD8 beta, can be expressed in the absence of CD8 alpha as a beta beta homodimer.

The T cell coreceptor CD8 exists on mature T cells as disulfide-linked homodimers of CD8 alpha polypeptide chains and heterodimers of CD8 alpha- and CD8 beta-chains. The function of the CD8 alpha-chain for binding to MHC class I and associating with the tyrosine kinase p56lck was demonstrated with CD8 alpha alpha homodimers. CD8 alpha beta functions as a better coreceptor, but the actual function of CD8 beta is less clear. Addressing this issue has been hampered by the apparent inability of CD8 beta to be expressed without CD8 alpha. This study demonstrates that human, but not mouse, CD8 beta can be expressed on the cell surface without CD8 alpha in both transfected COS-7 cells and murine lymphocytes. By creating chimeric proteins, we show that the murine Ig domain of CD8 beta is responsible for the lack of expression of murine CD8 beta beta dimers. In contrast to CD8 alpha alpha, CD8 beta beta is unable to bind MHC class I in a cell-cell adhesion assay. Detection of this form of CD8 should facilitate studies on the function of the CD8 beta-chain and indicates that caution should be used when interpreting studies on CD8 function using chimeric protein with the murine CD8 beta beta Ig domain. In addition, we demonstrate that the Ig domains of CD8 alpha are also involved in controlling the ability of CD8 to be expressed. Mutation of B- and F-strand cysteine residues in CD8 alpha reduced the ability of the protein to fold properly and, therefore, to be expressed.

Progressive expansion of an L-selectin-negative CD8 cell with anti-feline immunodeficiency virus (FIV) suppressor function in the circulation of FIV-infected cats.

The acute stage of feline immunodeficiency virus (FIV) infection is characterized by the appearance of a major CD8 subpopulation with reduced expression of the CD8 beta chain (CD8alpha+betalo). CD8 antiviral activity was subsequently shown to be mediated by the CD8alpha+betalo phenotype, which is the dominant CD8 phenotype in long-term infected cats. Two- and three-color flow cytometric analysis demonstrated that the CD8alpha+betalo subset is L-selectin negative (CD62L-) and has increased expression of CD44, CD49d, and CD18, consistent with an activation phenotype. The CD8alpha+betaloCD62L- cells but not the CD8alpha+betahiCD62L+ cells demonstrated strong antiviral activity in the FIV acute-infection assay. The progressive expansion of the CD8alpha+betaloCD62L- effector subset cells in FIV-infected cats parallels that seen in human immunodeficiency virus (HIV)-infected patients, suggesting that failure in homeostatic mechanisms regulating lymphocyte activation or trafficking (or both) may be a consequence of both HIV and FIV infections.

Early stages in the development of human T, natural killer and thymic dendritic cells.

T-cell development is initiated when CD34+ pluripotent stem cells or their immediate progeny leave the bone marrow to migrate to the thymus. Upon arrival in the thymus the stem cell progeny is not yet committed to the T-cell lineage as it has the capability to develop into T, natural killer (NK) and dendritic cells (DC). Primitive hematopoietic progenitor cells in the human thymus express CD34 and lack CD1a. When these progenitor cells develop into T cells they traverse a number of checkpoints. One early checkpoint is the induction of T-cell commitment, which correlates with appearance of CD1a and involves the loss of capacity to develop into NK cells and DC and the initiation of T-cell receptor (TCR) gene rearrangements. Basic helix-loop-helix transcription factors play a role in induction of T-cell commitment. CD1a+CD34+ cells develop into CD4+CD8 alpha+ beta+ cells by upregulating first CD4, followed by CD8 alpha and then CD8 beta. Selection for productive TCR beta gene rearrangements (beta selection) likely occurs in the CD4+CD8 alpha+ beta- and CD4+CD8 alpha+ beta+ populations. Although the T and NK-cell lineages are closely related to each other, NK cells can develop independently of the thymus. The fetal thymus is most likely one site of NK-cell development.

The CD8+ cell phenotype mediating antiviral activity in feline immunodeficiency virus-infected cats is characterized by reduced surface expression of the CD8 beta chain.

The acute stage of feline immunodeficiency virus (FIV) infection is characterized by a CD8+ anti-FIV response that parallels the appearance of a CD8+ subpopulation with reduced expression of the beta chain (CD8 alpha + beta lo). The relationship between the CD8 alpha + beta lo phenotype and CD8+ anti-FIV activity was examined. Flow cytometric analysis of peripheral blood mononuclear cells with anti-CD8 beta chain monoclonal antibody 117 revealed that the CD8 alpha + beta lo phenotype expanded throughout the asymptomatic infection, constituting 80%-90% of the CD8 beta + cells in long-term-infected cats. Purified CD8 alpha + beta hi and CD8 alpha + beta lo subpopulations were analyzed for anti-FIV activity in an acute infection assay. Anti-FIV activity resided principally in the CD8 alpha + beta lo population and was demonstrated in acute FIV infections, as well as in long-term asymptomatic infections. These data suggest that a unique CD8 alpha + beta lo anti-FIV phenotype arises early in infection and may play a major role in eliminating virus and maintaining the asymptomatic infection.

Co-capping studies reveal CD8/TCR interactions after capping CD8 beta polypeptides and intracellular associations of CD8 with p56(lck).

CD8 is a T cell surface glycoprotein that participates in recognition of peptide/MHC class I molecules by binding to their alpha 3 domains. In addition, the cytoplasmic domain of CD8 associates with the intracellular tyrosine kinase p56(lck) (lck) promoting recruitment of lck to the TCR signaling complex. Recent data have suggested also that CD8 may interact with the TCR to promote energetically favorable conformations which increase its ligand binding. We have used the techniques of co-capping and confocal microscopy to ask whether we can detect an association between CD8 and the TCR independently of their binding to MHC class I molecules. We show that capping CD8 heterodimers with antibodies to the CD8 beta polypeptide is significantly more efficient than antibodies to the CD8 alpha polypeptide at inducing co-localization of TCR molecules with CD8, suggesting that there may be preferred conformations of CD8 which stabilize interactions with the TCR. In addition, we show by microscopy that intracellular lck redistributes very efficiently to the area of a CD8 cap, suggesting that there is a stronger association between lck and CD8 than has been proposed from immunoprecipitation analyses.