Reduced immunosuppression in pediatric liver-intestine transplant recipients with CD8+CD28- T-suppressor cells

Twelve pediatric liver (n = 7), liver-kidney (n = 1), and small bowel (n = 4) transplant recipients, median age 6.5 years (1-21), who received rabbit anti-human thymocyte globulin (rATG) and steroid-free tacrolimus/sirolimus, were screened for the presence of CD8+CD28- T suppressor cells. Four control liver transplant recipients, median age 15 years (5-20), in whom conventional immunosuppression without rATG was successfully discontinued for at least 1 year, were also screened as a reference population. Median time to CD8+CD28- T-suppressor cells analysis was 16 months (2-24) in rATG subjects and 168 months (16-228) in no-immunosuppression subjects. Nine of 16 patients demonstrated the presence of CD8+CD28- T-suppressor cells in the circulation, whereas seven patients did not. CD8+CD28- T-suppressor cells were present in 4/4 children with no immunosuppression, and absent from three of four subjects with acute cellular rejection, all of whom experienced more than one acute cellular rejection episode. In the reduced immunosuppression group (n = 8), four children demonstrated presence of CD8+CD28- T-suppressor cells in the circulation and four did not. The presence of donor-specific T-suppressor cells in the circulation may characterize transplant recipients, in whom graft function can be maintained with minimal or no immunosuppression. Such assays may also permit safe evaluation of prospective immunosuppression withdrawal strategies.     

Generation of CD28- cells from long-term-stimulated CD8+CD28+ T cells: a possible mechanism accounting for the increased number of CD8+CD28- T cells in HIV-1-infected patients.

According to CD28 molecule expression, CD8+ T cells can be classed as CD28bright, CD28dim, and CD28-. The CD28dim T cells were found to derive from mitogenic stimulated CD28-T cells but also from CD28bright T cells through a mechanism of CD28 down-modulation. Moreover, after prolonged in vitro interleukin-2 stimulation, clonal CD28bright, cells showed a CD28dim expression before further evolution to a stable CD28-phenotype. This loss was concomitant with the disappearance of CD28 mRNA. A study of the cytokine production pattern revealed that CD28dim and CD28- T cell clones produced similar levels of type 1 and type 2 cytokines, which differed from those produced by the CD28bright T cell clones. A high percentage of CD28dim and CD28- cells, with similarities in their cytokine production pattern, were found in the blood samples of HIV-infected patients, as compared to healthy donors. The CD28 down-modulation may account for the increased number of CD8+CD28- T cells in HIV-infected patients.     

Regulatory CD8+CD28- T cells in heart transplant recipients

Human regulatory CD8+CD28- T cells (Ts) generated in vitro were demonstrated to suppress the activation and proliferation of T helper cells (Th) induced by allogeneic cells. This effect requires cell-to-cell contact, is antigen-specific, and results in Th anergy. To study the population of CD8+CD28- T cells present in vivo, flow cytometry was performed on whole blood specimens obtained from 25 heart transplant recipients and 12 normal controls. A significant expansion of CD8+CD28- T cells was found in transplant recipients as compared with normal individuals (p = 0.005). Expression of CD38, human leukocyte antigen-DR, and perforin positive cells within the CD8+CD28- subset was significantly higher in transplant patients than in normal controls, yet there was no correlation between the expression of these markers and acute rejection. Expression of the CD27 marker, however, was significantly higher within CD8+CD28- T cells from patients without rejection as compared with patients in rejection (p = 0.005), indicating that the memory-like CD8+CD28-CD27+ T-cell subset comprises regulatory cells, which play a protective role for the graft. CD8+CD28- T cells isolated from transplant patients did not display cytotoxic activity against donor cells and showed high expression of the killing inhibitory receptor CD94. This study identifies the phenotypic changes that occur in patients with heart transplants and opens new avenues for the induction of specific immunosuppression in transplantation.     

CD8+ CD28- and CD8+ CD57+ T cells and their role in health and disease

Chronic antigenic stimulation leads to gradual accumulation of late-differentiated, antigen-specific, oligoclonal T cells, particularly within the CD8(+) T-cell compartment. They are characterized by critically shortened telomeres, loss of CD28 and/or gain of CD57 expression and are defined as either CD8(+) CD28(-) or CD8(+) CD57(+) T lymphocytes. There is growing evidence that the CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell population plays a significant role in various diseases or conditions, associated with chronic immune activation such as cancer, chronic intracellular infections, chronic alcoholism, some chronic pulmonary diseases, autoimmune diseases, allogeneic transplantation, as well as has a great influence on age-related changes in the immune system status. CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell population is heterogeneous and composed of various functionally competing (cytotoxic and immunosuppressive) subsets thus the overall effect of CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell-mediated immunity depends on the predominance of a particular subset. Many articles claim that CD8(+) CD28(-) (CD8(+) CD57(+)) T cells have lost their proliferative capacity during process of replicative senescence triggered by repeated antigenic stimulation. However recent data indicate that CD8(+) CD28(-) (CD8(+) CD57(+)) T cells can transiently up-regulate telomerase activity and proliferate under certain stimulation conditions. Similarly, conflicting data is provided regarding CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell sensitivity to apoptosis, finally leading to the conclusion that this T-cell population is also heterogeneous in terms of its apoptotic potential. This review provides a comprehensive approach to the CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell population: we describe in detail its origins, molecular and functional characteristics, subsets, role in various diseases or conditions, associated with persistent antigenic stimulation.     

Foxp3 expressing CD4+ CD25+ and CD8+CD28- T regulatory cells in the peripheral blood of patients with lung cancer and pleural mesothelioma

The role of T regulatory (Treg) cells in human cancer has not yet been clarified. We assessed the presence and function of CD4+ and CD8+ Treg cell subsets in the peripheral blood of patients with lung cancer (LC) and pleural mesothelioma (PM). We found a low but significant increase in the number of CD4+ T cells with phenotype and functional features of Treg cells in LC patients compared to normal healthy controls (NHC). Furthermore, total CD4+ T cells from LC patients proliferated less than cells from controls, suggesting that the increase in the CD4+ Treg cell pool has functional importance. LC patients also showed an expansion of the CD8+CD28- T cell subset and these cells expressed Foxp3 mRNA, as recently observed in alloantigen-specific CD8+CD28- T suppressor cells. No variation of peripheral Treg cell subsets was found in patients with PM, a disease with a predominantly localized nature. However, the lack of correlation between cancer stage and the number or the function of peripheral Treg cells in LC patients refuted the hypothesis that these cells are involved in tumor spreading. A possible involvement of the peripheral Treg cell pool in cancer development and/or in inducing systemic immunosuppression in LC patients can be hypothesized.     

CD8+CD28-NRP-1+ T细胞在肾移植手术后患者外周血中的比例变化及意义

用流式细胞仪分别检测不同功能状态下肾移植术后患者体内的CD8~ CD28~-NRP-1~ Ts细胞和CD8~ CD28~-Foxp3~ Ts细胞,通过比较两类细胞的阳性表达率初步探讨它们之间的表达关系和意义。选取正常健康人10例,移植肾功能稳定的长期存活者24例,慢性移植物肾病者20例,急性排斥者15例,取受试者外周静脉血,分离单个核细胞,利用流式细胞仪检测CD8~ CD28~-NRP-1~ Ts细胞和CD8~ CD28~-Foxp3~ Ts细胞在CD8~ CD28~-T细胞群中的比例。结果表明组间比较得出,CD8~ CD28~-NRP-1~ Ts细胞和CD8~ CD28~-Foxp3~ Ts细胞比例在四组之间均有统计学差异(P<0.05)。其中在长期存活组表达量最高,其CD8~ CD28~-NRP-1~ Ts细胞和CD8~ CD28~-Foxp3~ Ts的比例分别为16.15%±1.49%和11.90%±2.73%,其次为正常健康人(13.83%±2.38%、9.44%±2.03%),然后为慢性移植物肾病组(8.03%±2.67%、5.26%±0.65%),而急排组最低(3.34%±1.73%、2.36%±1.14%),并且四组间这两种细胞呈同一变化趋势且前者的变化幅度大于后者。组内比较显示,CD8~ CD28~-NRP-1~ Ts细胞比例均高于CD8~ CD28~-Foxp3~ Ts细胞(P<0.05)。CD8~ CD28 NRP-1~ Ts细胞可以从整体水平上反映肾移植术后患者的免疫状态,它比CD8~ CD28~-Foxp3~ Ts细胞更能全面的反映术后患者的预后情况。     

CD8+CD28-NRP-1+ T细胞在肾移植手术后患者外周血中的比例变化及意义

用流式细胞仪分别检测不同功能状态下肾移植术后患者体内的CD8+CD28-NRP-1+ Ts细胞和CD8+CD28- Foxp3+ Ts细胞,通过比较两类细胞的阳性表达率初步探讨它们之间的表达关系和意义.选取正常健康人10例,移植肾功能稳定的长期存活者24例,慢性移植物肾病者20例,急性排斥者15例,取受试者外周静脉血,分离单个核细胞,利用流式细胞仪检测CD8+CD28-NRP-1+ Ts细胞和CD8+CD28- Foxp3+ Ts细胞在CD8+CD28-T细胞群中的比例.结果表明组间比较得出,CD8+CD28-NRP-1+ Ts细胞和CD8+CD28-Foxp3+ Ts细胞比例在四组之间均有统计学差异(P＜0.05).其中在长期存活组表达量最高,其CD8+CD28-NRP-1+ Ts细胞和CD8+CD28- Foxp3+ Ts的比例分别为16.15%±1.49%和11.90%±2.73%,其次为正常健康人(13.83%±2.38%、9.44%±2.03%),然后为慢性移植物肾病组(8.03%±2.67%、5.26%±0.65%),而急排组最低(3.34%±1.73%、2.36%±1.14%),并且四组间这两种细胞呈同一变化趋势且前者的变化幅度大于后者.组内比较显示,CD8+CD28-NRP-1+ Ts细胞比例均高于CD8+CD28- Foxp3+ Ts细胞(P＜0.05).CD8+CD28-NRP-1+ Ts细胞可以从整体水平上反映肾移植术后患者的免疫状态,它比CD8+CD28- Foxp3+ Ts细胞更能全面的反映术后患者的预后情况.     

Expansion of rare CD8+ CD28- CD11b- T cells with impaired effector functions in HIV-1-infected patients

The decline in the number of CD4+ T cells in HIV-1-infected patients is known to be related to the increased number of CD8+CD28- T cells. In this paper, we show that CD8+CD28- T cells from HIV-positive patients have an impaired capability to interact with human endothelial cells. This is due to the dramatic expansion, within this subset, of rare CD11b- cells lacking cell-cell adhesion functions. In 50 HIV-positive patients, 19.5% +/- 6.5% of all T cells were CD8+CD28-CD11b-, whereas only 0.8% +/- 0.4% of all T cells from healthy donors showed this uncommon phenotype. The percentage of circulating CD8+CD28-CD11b- T cells was strongly related to the percentage of CD4+ T cells (r = -0.82). This population is peculiar in terms of HIV infection and was found to possess some characteristics associated with effector functions but its cytotoxic properties were impaired. The percentage of target cells lysed by CD8+CD28-CD11b- was significantly lower than that of cells lysed by its CD11b- counterpart (p <.05) both at low (5:1) or at relatively high (20:1) effector/target ratios. CD8+CD28-CD11b- T cells, which lack the ability to interact with endothelial cells, are likely to accumulate and persist in circulation. The biologic properties of CD8+CD28-CD11b- T cells suggest that these cells might be endstage or aberrant differentiated effector cells. Lack of cell-cell adhesion and impaired cytolytic functions favor the hypothesis of a role for CD8+CD28-CD11b- T cells in the development of immunodeficiency.     

Altered ex vivo balance between CD28+ and CD28− cells within HIV-specific CD8+ T cells of HIV-seropositive patients

The CD8⁺ CD28⁻ cell population in the blood of HIV-infected individuals is considerably expanded. Yet the cause of this expansion is not clear. The recent demonstration of identical TCR-rearranged genes in CD8⁺ CD28⁺ and CD8⁺ CD28⁻ expanded T cells of HIV-seropositive patients supports the hypothesis that these two subpopulations are phenotypic variants of the same lineage. To further elucidate the precise relationship between them, we measured the fraction of CD28⁺ and CD28⁻ T cell subsets in IFN-γ-producing CD8⁺ T cells by intracellular staining and cytofluorometry as a functional test for ex vivo recognition of epitopes derived from HIV-1, Epstein-Barr virus (EBV) and influenza virus. HIV-specific CD8⁺ T cells were predominantly CD28⁻ in all the eight HIV-seropositive subjects tested. In contrast, the anti-EBV and anti-influenza CD8⁺ T cells were mainly CD28⁺ in these patients as well as in HIV-seronegative individuals. This supports the notion that the CD8⁺ CD28⁻ hyperlymphocytosis observed in HIV infection is due mainly to chronic activation and differentiation of HIV-specific memory CD8⁺ CD28⁺ T cells into terminally differentiated CD8⁺ CD28⁻ lymphocytes, because of intense HIV-1 replication and without any important bystander activation. This clarification of the mechanisms underlying the CD8⁺ CD28⁻ expansion in HIV-induced pathogenesis may have important therapeutic implications.     

Differentiation of human CD8 T cells: implications for in vivo persistence of CD8+ CD28- cytotoxic effector clones

CD8 T cells contain a distinct subset of CD8+ CD28- cells. These cells are not present at birth and their frequency increases with age. They frequently contain expanded clones using various TCRalphabeta receptors and these clones can represent >50% of all CD8 cells, specially in old subjects or patients with chronic viral infections such as HIV-1. Herein, it is shown that a large fraction of CD8+ CD28- cells expresses intracellular perforin by three-color flow cytometry, in particular when this subset is expanded. Together with their known ability to exert potent re-directed cytotoxicity, this indicates that CD8+ CD28- T cells comprise cytotoxic effector cells. With BrdU labeling, we show that CD8+ CD28- cells derive from CD8+ CD28+ precursors in vitro. In addition, sorted CD8+ CD28+ cells gave rise to a population of CD8+ CD28- cells after allo-stimulation. Moreover, ex vivo CD8+ CD28+ cells contain the majority of CD8 blasts, supporting the notion that they contain the proliferative precursors of CD8+ CD28- cells. CD95 (Fas) expression was lower in CD8+ CD28- cells, and this subset was less prone to spontaneous apoptosis in ex vivo samples and more resistant to activation-induced cell death induced by a superantigen in vitro. Thus, the persistence of expanded clones in vivo in the CD8+ CD28- subset may be explained by antigen-driven differentiation from CD8+ CD28+ memory precursors, with relative resistance to apoptosis as the clones become perforin(+) effector cells.     

CD8+ T cells and not CD4+ T cells are hyporesponsive to CD28- and CD40L-mediated activation in HIV-infected subjects

T cell dysfunction in HIV-infected subjects could be the consequence of altered sensitivity of CD4⁺ or CD8⁺ T cells to various costimulatory signals. Therefore, we studied proliferation and cytokine production in highly purified CD8⁺ and CD4⁺ T cells from HIV-infected and HIV⁻ subjects, induced by co-activation via cell-bound CD80, CD86 and CD40 or by allo-activation. Regardless of the nature of the first and the costimulatory signal, CD8⁺ T cells from patients proliferated consistently less than controls, while responses from CD4⁺ T cells were similar in patients and controls. This phenomenon was observed after ligation of CD28 combined with anti-CD3 or phorbol myristate acetate (PMA), but also after allogeneic stimulation and after activation by CD40 and anti-CD3. Anti-CD3 combined with CD80 or CD86 induced a mixed Th1/Th2-type cytokine profile in both CD4⁺ and CD8⁺ T cells from controls, whereas anti-CD3 plus CD40 induced only low levels of Th2-type cytokines and no interferon-gamma (IFN-γ) in CD4⁺ T cells. Compared with controls, CD4⁺ T cells from patients produced slightly lower levels of IL-10 but equal amounts of IFN-γ, IL-4 and IL-5, while CD8⁺ T cells from patients produced less of all cytokines tested. In conclusion, responses of purified CD4⁺ T cells from HIV⁺ subjects to various costimulatory pathways are relatively intact, whereas CD8⁺ T cells are hyporesponsive at the level of proliferation and cytokine production. A generalized intrinsic CD8⁺ T cell failure might contribute to viral and neoplastic complications of HIV infection.     

胃癌患者PBMCs中CD4+CD25+T细胞体外增殖及对CD4+CD25-T细胞增殖的影响

目的:探讨胃癌患者外周血单个核细胞（PBMCs）中的CD4+CD25+T细胞体外增殖及对CD4+CD25-T细胞增殖的影响。 方法:以免疫磁性分离方法 （MACS）分选出胃癌患者外周血单个核细胞中的CD4+CD25+T及CD4+CD25-T细胞后，用流式细胞仪分析细胞的纯度及活力；再以小鼠抗人CD3单抗、小鼠抗人CD28单抗及rh IL-2作为共刺激因子，观察与CD4+CD25-T细胞共培养时，CD4+CD25+T细胞对CD4+CD25-T细胞增殖的抑制效应。 结果:（1）分选后健康对照组及胃癌患者PBMC 中CD4+CD25+ T细胞纯度分别为83.8%±1.84%、84.13%±2.77％，两者相比，无显著差异（P＞0.05）;(2)经MACS 分选后正常对照组与胃癌患者CD4+CD25+ T细胞活力分别为98.52%±0.72%、97.80%±0.95%，两者相比，无显著差异（P＞0.05）；(3)无论是健康对照还是胃癌患者的CD4+CD25+T均具有明显抑制效应性T细胞如CD4+CD25-T细胞的增殖，随着CD4+CD25+T细胞数的增加，这种抑制增殖的能力也相应增加，当CD4+CD25+∶〖KG-*2〗CD4+CD25-T达 1∶〖KG-*2〗1时，抑制率最大达到50％。 结论:MACS分选法能够分选出高纯度及活力的CD4+CD25+T细胞，分选后CD4+CD25+T细胞在体外均能抑制CD4+CD25-T细胞增殖，且这种抑制效应呈一定效靶比关系。     

Expansion of cytotoxic CD8+ CD28- T cells in healthy ageing people, including centenarians.

Ageing is associated with complex remodelling in the phenotypic and functional profiles of T lymphocytes. We investigated whether expression of CD28 antigen on T cells is conserved throughout adulthood and ageing in humans. For this purpose we analysed T cells obtained from peripheral blood of 102 healthy people of ages ranging from 20 to 105 years. We found an age-related increase of CD28- T cells in percentage and absolute number, predominantly among CD8+ T cells. CD28- T cells from aged donors analysed by flow cytometry appeared as resting cells (not expressing CD25, CD38, CD69, CD71, DR), bearing markers of cytotoxic activity (CD 11b and CD 57) and with a phenotype compatible with 'memory' cells (up-regulated CD2 and CD11a; CD62L absent). At the functional level, freshly isolated purified CD28- CD8+ T cells showed high anti-CD3 redirected cytotoxic activity against Fc-bearing P815 cells. The same activity tested on freshly isolated bulk T lymphocytes was significantly augmented with age. We found a positive correlation between age, number of CD8+ CD28- T cells and anti-CD3 redirected cytotoxicity by freshly isolated T cells. These data suggest that an activation of unknown nature within the cytotoxic arm of the immune system occurs with age. We speculate that these cytotoxic T lymphocytes (CTL) in vivo may constitute armed effector cells for immediate killing of targets bearing peptides from pathogens of intracellular origin.     

DNA甲基化在急性冠脉综合征患者CD4+CD28-T细胞CD28表达缺失中的作用

目的 通过研究急性冠脉综合征(acute coronary syndrome,ACS)患者外周血CD4+T细胞CD28 mRNA水平和CD28基因启动子调节序列的甲基化状态,旨在探讨DNA甲基化在ACS患者CD4+CD28-T细胞CD28表达缺失中的作用.方法 免疫磁珠分离CD4+T细胞经逆转录后,实时定量PCR(real time-PCR)技术检测CD4+T细胞CD28mRNA的表达水平,亚硫酸氢钠测序检测CD28基因启动子调节序列的甲基化状态.结果 与正常对照组相比,ACS患者CD4+T细胞CD28mRNA表达水平显著减低,差异具有统计学意义[正常对照组比ACS组:(1.066±0.162)比(0.401±0.069),P<0.05].CD28基因启动子区域的甲基化水平显著增高,差异有统计学意义[正常对照组比ACS组:(24.47±3.17)%比(43.33±1.52)%,P<0.05].CD28基因启动子区域DNA甲基化水平与CD28mRNA表达水平呈显著负相关(P=0.01,r=-0.579).结论 ACS患者CD4+T细胞CD28基因启动子区域高甲基化调控了CD28基因转录抑制.DNA甲基化参与了CD4+CD28-T细胞的形成.     

CD8+ CD28− T cells in vertically HIV-infected children

To evaluate whether vertical HIV infection interferes with the expression of CD28 on T lymphocytes, 25 HIV-infected children and 29 seroreverted children born to HIV⁺ mothers were studied. The percentage of CD28⁻ cells among CD8⁺ T lymphocytes was higher in HIV-infected children than in controls (P < 0.001). In fact, in HIV-infected children, this percentage was elevated from the first year of life, while in healthy seroreverted children, the proportion of CD28⁻ cells among CD8⁺ cells rose progressively with age (r = 0.49; P = 0.008). In HIV⁺ children, the CD8⁺ CD28⁻, but not CD8⁺ CD28⁺ cell proportion was significantly correlated with immunological markers of disease progression, such as CD4⁺ cell loss (r = −0.65; P < 0.001) and the level of in vitro spontaneous lymphocyte apoptosis (r = 0.53; P = 0.03).     

Nonantigen specific CD8+ T suppressor lymphocytes originate from CD8+CD28- T cells and inhibit both T-cell proliferation and CTL function

Nonantigen specific CD8+ suppressor T lymphocytes (CD8+ Ts) inhibit T-cell proliferation of antigen-specific T lymphocytes. The impossibility to generate in vitro these cells has been correlated with the appearance of relapses in patients affected by autoimmune diseases, suggesting the involvement of these cells in immune regulation. This study was aimed to identify circulating precursors and to characterize the phenotype and mechanism of action of CD8+ Ts. We found that CD8+ Ts can be generated in vitro from CD8+CD28- T lymphocytes, but not from CD8+CD28+ T cells. A key role in their generation is played by monocytes that secrete interleukin-10 (IL-10) after granulocyte macrophage-colony-stimulating factor (GM-CSF) stimulation. Cell-to-cell direct interaction between CD8+CD28- T cells and monocytes does not play a role in the generation of CD8+ Ts. CD8+ Ts have a CD45RA+, CD27-, CCR7-, IL-10Ralpha+ phenotype and a TCR Vbeta chain repertoire overlapping that of autologous circulating CD8+ T cells. This phenotype is typical of T lymphocytes previously expanded due to antigen stimulation. Their suppressive effect on T-cell proliferation targets both antigen presenting cells, such as dendritic cells, and antigen-specific T lymphocytes, and is mediated by IL-10. CD8+ Ts suppress also the antigen-specific cytotoxic activity of CTL decreasing the expression of HLA class I molecules on target cells through IL-10 secretion. These findings can be helpful for the better understanding of immune regulatory circuits and for the definition of new pathogenic aspects in autoimmunity and tumor immunology.     

Mesenchymal stem cells control alloreactive CD8+CD28− T cells

CD28/B7 co-stimulation blockade with belatacept prevents alloreactivity in kidney transplant patients. However, cells lacking CD28 are not susceptible to belatacept treatment. As CD8⁺CD28⁻ T-cells have cytotoxic and pathogenic properties, we investigated whether mesenchymal stem cells (MSC) are effective in controlling these cells. In mixed lymphocyte reactions (MLR), MSC and belatacept inhibited peripheral blood mononuclear cell (PBMC) proliferation in a dose-dependent manner. MSC at MSC/effector cell ratios of 1:160 and 1:2·5 reduced proliferation by 38·8 and 92·2%, respectively. Belatacept concentrations of 0·1 μg/ml and 10 μg/ml suppressed proliferation by 20·7 and 80·6%, respectively. Both treatments in combination did not inhibit each other''s function. Allostimulated CD8⁺CD28⁻ T cells were able to proliferate and expressed the cytolytic and cytotoxic effector molecules granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. While belatacept did not affect the proliferation of CD8⁺CD28⁻ T cells, MSC reduced the percentage of CD28⁻ T cells in the proliferating CD8⁺ T cell fraction by 45·9% (P = 0·009). CD8⁺CD28⁻ T cells as effector cells in MLR in the presence of CD4⁺ T cell help gained CD28 expression, an effect independent of MSC. In contrast, allostimulated CD28⁺ T cells did not lose CD28 expression in MLR–MSC co-culture, suggesting that MSC control pre-existing CD28⁻ T cells and not newly induced CD28⁻ T cells. In conclusion, alloreactive CD8⁺CD28⁻ T cells that remain unaffected by belatacept treatment are inhibited by MSC. This study indicates the potential of an MSC–belatacept combination therapy to control alloreactivity.     

Proliferative arrest and cell cycle regulation in CD8(+)CD28(-) versus CD8(+)CD28(+) T cells

CD8(+)CD28(-) T cells have been characterized by oligoclonal expansions, impaired proliferative responses, but preserved cytotoxicity and reduced telomeres. To examine this subset further and define the underlying mechanisms of proliferation arrest, we investigated several features of this cell type compared with CD8(+)CD28(+) controls. We analyzed expression of various activation markers, thymidine incorporation upon activation, T-cell receptor (TCR) zeta-chain phosphorylation, cell cycle characteristics, and cell cycle related gene expression. Flow cytometry revealed higher expression of CD11b, CD29, CD57, and CD94, and lower expression of CD25 in CD8(+)CD28(-) compared with CD8(+)CD28(+) T cells. Sorted CD8(+)CD16(-)CD28(-) cells exhibited decreased phosphorylation of the TCR zeta-chain in three of four probands. Proliferation of these T cells was impaired, even when activated with mitogens that bypass TCR signaling. Cell cycle profiles demonstrated a lower percentage of cycling cells and significantly higher levels of cyclin dependent kinase inhibitor p16(INK4a) in the CD28(-) subset compared with the CD28(+) control. These observations suggest that expanded CD8(+)CD28(-) T cells in normal elderly individuals have reduced proliferation concomitant with increased p16(INK4a) expression. Defects in TCR signaling were associated with altered TCR zeta-chain phosphorylation.     

TCR repertoire of suppressor CD8+CD28- T cell populations.

The cellular basis of graft rejection and the development of strategies for specific suppression of T cell responses against allogeneic and xenogeneic transplants represents an area of active investigation. Recently, a population of MHC-class I restricted CD8+CD28- T suppressor cells (Ts) which are able to inhibit specifically the proliferative response of allospecific, xenospecific and nominal-antigen specific CD4+ T helper cells (Th) has been identified. We have studied the TCR V beta gene repertoire expressed by CD8+CD28- Ts isolated from allospecific, xenospecific, and nominal antigen-specific T cell lines (TCL). A limited V beta repertoire has been found in all TCLs studied. The most restricted TCR V beta usage was observed within the population of Ts from xenospecific TCLs. The TCR V beta usage within the Ts subset of TCL differs from the TCR repertoire expressed by the CD4+ Th subset of the same TCL. This is consistent with the fact that Ts and Th cells recognize distinct MHC/ antigen complexes. The finding that the TCR repertoire used by Ts is limited opens new avenues for studying the mechanisms of transplant rejection.