Quantitative analysis of T cell homeostatic proliferation

T cell homeostatic proliferation occurs on transfer of T cells into lymphopenic recipients; transferred cells undergo several rounds of division in the absence of specific antigen stimulation. For a quantitative analysis of this phenomenon, we applied a mathematical method to describe proliferating T cells to match peak distributions from actual CFSE dilution data. For in vitro stimulation of T cells with anti-CD3/anti-CD28, our simulation confirmed a high proportion of cells entering cell cycle with a low proportion undergoing apoptosis. When applied to homeostatic proliferation, it described striking differences in CD4 and CD8 T cell proliferation rates, and accurately predicted that successive divisions were accompanied by higher rates of apoptosis, limiting the accumulation of proliferating cells. Thus, the presence of multiple CFSE dilution peaks cannot be considered equivalent to lymphocyte expansion. Finally, genetic effects were identified that may help explain links between homeostatic proliferation and autoimmunity.     

B cell depletion enhances T regulatory cell activity essential in the suppression of arthritis

The efficacy of B cell-depletion therapy in rheumatoid arthritis has driven interest in understanding the mechanism. Because the decrease in autoantibodies in rheumatoid arthritis does not necessarily correlate with clinical outcome, other mechanisms may be operative. We previously reported that in proteoglycan-induced arthritis (PGIA), B cell-depletion inhibits autoreactive T cell responses. Recent studies in B cell-depletion therapy also indicate a role for B cells in suppressing regulatory mechanisms. In this study, we demonstrate that B cells inhibited both the expansion and function of T regulatory (Treg) cells in PGIA. Using an anti-CD20 mAb, we depleted B cells from mice with PGIA and assessed the Treg cell population. Compared to control Ab-treated mice, Treg cell percentages were elevated in B cell-depleted mice, with a higher proportion of CD4(+) T cells expressing Foxp3 and CD25. On a per-cell basis, CD4(+)CD25(+) cells from B cell-depleted mice expressed increased amounts of Foxp3 and were significantly more suppressive than those from control Ab-treated mice. The depletion of Treg cells with an anti-CD25 mAb concurrent with B cell-depletion therapy restored the severity of PGIA to levels equal to untreated mice. Although titers of autoantibodies did not recover to untreated levels, CD4(+) T cell recall responses to the immunizing Ag returned as measured by T cell proliferation and cytokine production. Thus, B cells have the capacity to regulate inflammatory responses by enhancing effector T cells along with suppressing Treg cells.     

Natural CD825 regulatory T cell-secreted exosomes capable of suppressing cytotoxic T lymphocyte-mediated immunity against B16 melanoma

Natural CD4⁺25⁺ and CD8⁺25⁺ regulatory T (Tr) cells have been shown to inhibit autoimmune diseases. Immune cells secrete exosomes (EXOs), which are crucial for immune regulation. However, immunomodulatory effect of natural Tr cell-secreted EXOs is unknown. In this study, we purified natural CD8⁺25⁺ Tr cells from C57BL/6 mouse naive CD8⁺ T cells, and in vitro amplified them with CD3/CD28 beads. EXOs (EXO_Tr) were purified from Tr cell’s culture supernatants by differential ultracentrifugation and analyzed by electron microscopy, Western blot and flow cytometry. Our data showed that EXO_Tr had a “saucer” or round shape with 50–100 nm in diameter, contained EXO-associated markers LAMP-1 and CD9, and expressed natural Tr cell markers CD25 and GITR. To assess immunomodulatory effect, we i.v. immunized C57BL/6 mice with ovalbumin (OVA)-pulsed DCs (DC_OVA) plus Tr cells or EXO_Tr, and then assessed OVA-specific CD8⁺ T cell responses using PE-H-2K^b/OVA tetramer and FITC-anti-CD8 antibody staining by flow cytometry and antitumor immunity in immunized mice with challenge of OVA-expressing BL6–10_OVA melanoma cells. We demonstrated that DC_OVA-stimulated CD8⁺ T cell responses and protective antitumor immunity significantly dropped from 2.52% to 1.08% and 1.81% (p < 0.05), and from 8/8 to 2/8 and 5/8 mice DC_OVA (p < 0.05) in immunized mice with co-injection of Tr cells and EXO_Tr, respectively. Our results indicate that natural CD8⁺25⁺ Tr cell-released EXOs, alike CD8⁺25⁺ Tr cells, can inhibit CD8⁺ T cell responses and antitumor immunity. Therefore, EXOs derived from natural CD4⁺25⁺ and CD8⁺25⁺ Tr cells may become an alternative for immunotherapy of autoimmune diseases.     

A regulatory role for CD37 in T cell proliferation

CD37 is a leukocyte-specific protein belonging to the tetraspanin superfamily. Previously thought to be predominantly a B cell molecule, CD37 is shown in this study to regulate T cell proliferation. CD37-deficient (CD37(-/-)) T cells were notably hyperproliferative in MLR, in response to Con A, or CD3-TCR engagement particularly in the absence of CD28 costimulation. Hyperproliferation was not due to differences in memory to naive T cell ratios in CD37(-/-) mice, apoptosis, or TCR down-modulation. Division cycle analyses revealed CD37(-/-) T cells to enter first division earlier than wild-type T cells. Importantly, proliferation of CD37(-/-) T cells was preceded by enhanced early IL-2 production. We hypothesized CD37 to be involved in TCR signaling and this was supported by the observation that CD4/CD8-associated p56(Lck) kinase activity was increased in CD37(-/-) T cells. Remarkably, CD37 cross-linking on human T cells transduced signals that led to complete inhibition of CD3-induced proliferation. In the presence of CD28 costimulation, CD37 engagement still significantly reduced proliferation. Taken together, these results demonstrate a regulatory role for CD37 in T cell proliferation by influencing early events of TCR signaling.     

The role of alpha beta+ T cells and homeostatic T cell proliferation in Y-chromosome-associated murine lupus

Male BXSB mice develop an early life, severe lupus-like disease largely attributed to an undefined Y-chromosome-associated autoimmunity accelerator, termed YAA: Although the exact disease pathogenesis is uncertain, indirect evidence suggests that T cells play an important role in the male BXSB disease. We have developed TCR alpha-chain gene-deleted BXSB mice to directly examine the role of alphabeta+ T cells and the mode by which Yaa promotes disease in this strain. All disease parameters, including hypergammaglobulinemia, autoantibody production, glomerulonephritis, and the unique monocytosis of BXSB males, were severely reduced or absent in the alphabeta+ T cell-deficient mice. Adoptively transferred CD4+ T cells of either male or female BXSB origin showed equal homeostatic proliferation in alphabeta+ T cell-deficient male recipients. Moreover, deficient male mice eventually developed equally severe lupus-like disease after adoptive transfer and homeostatic expansion of T cells from wild-type BXSB males or females. The results directly demonstrate that the Yaa-mediated disease requires alphabeta+ T cells that are not, in themselves, abnormal in either composition or properties, but are engaged by a Yaa-encoded abnormality in a non-T cell component. In addition, homeostatic anti-self proliferation of mature T cells derived from a small number of precursors can induce systemic autoimmunity in an appropriate background.     

Global natural regulatory T cell depletion in active systemic lupus erythematosus

The immune defect that could account for the multisystemic involvement that characterizes systemic lupus erythematosus (SLE) remains unknown. We hypothesized that iterative disease flares correspond to a recurrent defect in the peripheral immune suppression exerted by naturally occurring T regulatory cells (Tregs). Surprisingly, Tregs isolated from lupus patients show the same phenotypic and functional characteristics as corresponding cells found in healthy controls. A decrease in the proportion of circulating Tregs among other CD4+ T cells is nevertheless evidenced in active patients when this group is compared with healthy controls (0.57 +/- 0.24%, n = 45 vs 1.29 +/- 0.38%, n = 82, p < 0.0001) or with inactive patients (1.22 +/- 0.67%, n = 62, p < 0.0001). In contrast, the proportion of Tregs in other systemic autoimmune diseases such as primary Sj?gren syndrome and inflammatory myopathy does not significantly differ from controls' values (1.15 +/- 0.46%, n = 21, p = 0.09 and 1.16 +/- 0.44%, n = 16, p = 0.43, respectively). Lupus Tregs do not accumulate in either the lymph nodes or the diseased kidneys and are not killed by a circulating soluble factor, but demonstrate in vitro a heightened sensitivity to Fas-induced apoptosis. Finally, we show that the extent of Treg depletion correlates with the clinical severity of the flare. SLE flares are therefore associated with a global Treg depletion and not with a phenomenon of tissue redistribution. In summary, we suggest that the physiopathology of SLE could be tied to a defect in the homeostatic control of the Treg subpopulation.     

Dehydroleucodine and dehydroparishin-B inhibit proliferation and motility of B16 melanoma cells

Dehydroleucodine, a known sesquiterpene lactone, and dehydroparishin-B, a new guaiane type sesquiterpene acid, were isolated from aerial parts of Artemisia douglasiana by chloroform extraction. We identified dehydroparishin-B as (7R)-2-oxo-guaia-1(10),3(4),5(6),11(13)-tetraen-12-oic acid by MS and NMR methods. We demonstrated that both dehydroparishin-B and dehydroleucodine blocked cell proliferation of B16 melanoma cells, but not normal murine Melan-A melanocytes, in a dose-dependent manner without affecting cell viability. We also found that both dehydroparishin-B and dehydroleucodine inhibited migration of B16 melanoma cells. These results suggest that dehydroleucodine and dehydroparishin-B could represent potential candidates for the treatment of metastatic melanomas.     

Interferon-alpha-induced inhibition of B16 melanoma cell proliferation: interference with the bFGF autocrine growth circuit.

The molecular mechanisms underlying the growth inhibition induced by interferon-alpha (IFN-alpha) in B16 murine melanoma cells were investigated. IFN-alpha did not induce cell apoptosis, but strongly interfered with the synthesis of basic fibroblast growth factor (bFGF), which acts as an autocrine growth factor in this system. Inhibition of bFGF synthesis was observed at the same concentrations (50-500 pM, 10-100 U/ml) of IFN-alpha able to induce growth arrest of B16 melanoma cells. Although the synthesis of acidic (a)FGF was only slightly affected by IFN-alpha, the cytokine induced release of an aFGF-related low-molecular-weight peptide, which was able to interfere with bFGF binding to surface receptors. Thus, the molecular mechanisms of IFN-alpha activity on melanoma cells include a specific modulation of the bFGF autocrine circuit.     

Infiltration of a mesothelioma by IFN-gamma-producing cells and tumor rejection after depletion of regulatory T cells

Depletion of CD4+CD25+Foxp3+ regulatory T cells (CD25+ T(reg)) with an anti-CD25 Ab results in immune-mediated rejection of tolerogenic solid tumors. In this study, we have examined the immune response to a mesothelioma tumor in mice after depletion of CD25+ cells to elucidate the cellular mechanisms of CD25+ T(reg), a subject over which there is currently much conjecture. Tumor rejection was found to be primarily due to the action of CD8+ T cells, although CD4+ cells appeared to play some role. Depletion of CD25+ cells resulted in an accumulation in tumor tissue of CD4+ and CD8+ T cells and NK cells that were producing the potent antitumor cytokine IFN-gamma. Invasion of tumors by CD8+ T cells was partially dependent on the presence of CD4+ T cells. Although a significant increase in the proliferation and number of tumor-specific CD8+ T cells was observed in lymph nodes draining the tumor of anti-CD25-treated mice, this effect was relatively modest compared with the large increase in IFN-gamma-producing T cells found in tumor tissue, which suggests that the migration of T cells into tumor tissue may also have been altered. Depletion of CD25+ cells did not appear to modulate antitumor CTL activity on a per cell basis. Our data suggests that CD25+ T(reg) limit the accumulation of activated T cells producing IFN-gamma in the tumor tissue and, to a lesser extent, activation and/or rate of mitosis of tumor-specific T cells in lymph nodes.     

Accessory cell function of human melanoma cells in mitogen-induced T cell proliferation

Six out of eight human melanoma cell lines were found to be able to function as accessory cells in PHA-induced proliferation of autologous and allogeneic T cells. The accessory cell function of the melanoma cell lines appears to be similar to that of monocytes, requires the presence of viable cells, and does not correlate with the cell surface binding sites for PHA and with the level of expression of HMW-MAA and of HLA Class I antigens. HLA Class II antigens do not appear to play a major role in these phenomena, since there is no relationship between level of expression of HLA Class II antigens and accessory cell function of melanoma cells. Furthermore, addition of anti-HLA Class II monoclonal antibodies does not affect proliferation of T cells stimulated with PHA in the presence of melanoma cells with accessory cell function. Although melanoma cells exert accessory cell function, functional and immunological assays did not detect IL-1 in the spent medium of the melanoma cell lines. Furthermore, Northern blotting analysis with IL-1 alpha and IL-1 beta probes did not detect IL-1-specific mRNA in melanoma cell lines. These results suggest that PHA-induced proliferation of T cells in the presence of melanoma cells can bypass the requirement for IL-1 or utilizes factors other than IL-1.     

Fulfilling the metabolic requirements for cell proliferation

The activity of key metabolic enzymes is regulated by the ubiquitin ligases that control the function of the cyclins; therefore the activity of these ubiquitin ligases explains the coordination of cell-cycle progression with the supply of substrates necessary for cell duplication. APC/C (anaphase-promoting complex/cyclosome)-Cdh1, the ubiquitin ligase that controls G1- to S-phase transition by targeting specific degradation motifs in cell-cycle proteins, also regulates the glycolysis-promoting enzyme PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3) and GLS1 (glutaminase 1), a critical enzyme in glutaminolysis. A decrease in the activity of APC/C-Cdh1?in mid-to-late G1 releases both proteins, thus explaining the simultaneous increase in the utilization of glucose and glutamine during cell proliferation. This occurs at a time consistent with the point in G1 that has been described as the nutrient-sensitive restriction point and is responsible for the transition from G1 to S. PFKFB3 is also a substrate at the onset of S-phase for the ubiquitin ligase SCF (Skp1/cullin/F-box)-β-TrCP (β-transducin repeat-containing protein), so that the activity of PFKFB3 is short-lasting, coinciding with a peak in glycolysis in mid-to-late G1, whereas the activity of GLS1 remains high throughout S-phase. The differential regulation of the activity of these proteins indicates that a finely-tuned set of mechanisms is activated to fulfil specific metabolic demands at different stages of the cell cycle. These findings have implications for the understanding of cell proliferation in general and, in particular, of cancer, its prevention and treatment.     

A closer look at homeostatic proliferation of CD4+ T cells: costimulatory requirements and role in memory formation

Ag-specific proliferation of CD4+ T cells is regulated, in part, by costimulatory signals through CD28. The proliferative response during primary activation is an important determinant of the ability of the T cell to respond to Ag re-encounter. Proliferation of mature CD4+ T cells during lymphopenia (homeostatic proliferation) requires interaction with endogenous peptide MHC. However, the role of costimulation during homeostatic proliferation is unclear, as is the ability of homeostatic proliferation to regulate secondary T cell responses. Using a TCR transgenic system and serial adoptive transfers we find that homeostatic proliferation of CD4+ T cells occurs for at least 5 wk after adoptive transfer into recombination-activating gene (RAG)-/- recipients. Two discrete populations of proliferating T cells can be resolved, one that is highly proliferative and dependent on CD28 signaling, and the other that contains cells undergoing low levels of CD28-independent proliferation. Importantly, naive CD4+ T cells that have undergone homeostatic proliferation acquire both phenotypic and functional characteristics of true memory cells. These studies indicate that functional memory T cells can be generated by encounters with endogenous Ags only. This mechanism of T cell regeneration is possibly active during lymphopenia due to viral infections, such as HIV, transplantation, or cancer therapy, and may explain selected autoimmune diseases.     

The molecular mechanisms of vitamin C on cell cycle regulation in B16F10 murine melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose-dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53-p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C-induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53-p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K-family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C-induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2-p53-p21Waf1/Cip1 pathway.     

T-cadherin suppresses the cell proliferation of mouse melanoma B16F10 and tumor angiogenesis in the model of the chorioallantoic membrane

The influence of T-cadherin on the pigmentation and proliferation of mouse melanoma B16F10 cells in vitro and on the growth and neovascularization of tumor cell masses formed by the B16F10 cells in a model of the chorioallantoic membrane of a chicken embryo is studied. It is found that the proliferative activity of the cells decreases in the cell culture of mouse melanoma upon the overexpression of T-cadherin in comparison with the cells in the control. It is shown in experiments in vitro that the B16F10 cells with the overexpression of T-cadherin are rarely settling are to the chorioallantoic membrane than the control cells. In addition, it is found that the control cells of mouse melanoma form tumors with area more 0.1 mm² more often than the cells with the overexpression of T-cadherin and the amount of the vessels growing to tumor cell masses formed by the cells with the overexpression of T-cadherin is significantly lower than the same index for the cells in the control. Thus, the overexpression of T-cadherin in the B16F10 cells suppresses the proliferation of these cells in vitro and the growth of the tumor masses formed by melanoma cells on the chorioallantoic membrane and their neovascularization in vivo.     

Lymphopenia-induced homeostatic proliferation of CD8+ T cells is a mechanism for effective allogeneic skin graft rejection following burn injury

Burn patients are immunocompromised yet paradoxically are able to effectively reject allogeneic skin grafts. Failure to close a massive burn wound leads to sepsis and multiple system organ failure. Immune suppression early (3 days) after burn injury is associated with glucocorticoid-mediated T cell apoptosis and anti-inflammatory cytokine responses. Using a mouse model of burn injury, we show CD8+ T cell hyperresponsiveness late (14 days) after burn injury. This is associated with a CD8+ T cell pro- and anti-inflammatory cytokine secretion profile, peripheral lymphopenia, and accumulation of a rapidly cycling, hyperresponsive memory-like CD8+CD44+ IL-7R- T cells which do not require costimulation for effective Ag response. Adoptive transfer of allospecific CD8+ T cells purified 14 days postburn results in enhanced allogeneic skin graft rejection in unburned recipient mice. Chemical blockade of glucocorticoid-induced lymphocyte apoptosis early after burn injury abolishes both the late homeostatic accumulation of CD8+ memory-like T cells and the associated enhanced proinflammatory CD8+ T cell response, but not the late enhanced CD8+ anti-inflammatory response. These data suggest a mechanism for the dynamic CD8+ T cell response following injury involving an interaction between activation, apoptosis, and cellular regeneration with broad clinical implications for allogeneic skin grafting and sepsis.     

Effect of serum withdrawal on the proliferation of B16F10 melanoma cells

B16F10 murine melanoma cell proliferation was inhibited after 48 h in medium with serum in the range 0.1 to 0.5% by volume. Cell viability was mostly retained, whereas cells completely deprived of serum died. Growth-arrested cultures showed serum-dependent suppression of DNA synthesis. The response was typically that of a 'cell cycle freeze', verified by flow cytometric distribution of cells. Consequently, serum deprivation did not lead to synchrony when serum was restored to arrested populations. Furthermore, there was no change in PCNA expression in arrested cells.     

Partial regulatory T cell depletion prior to schistosomiasis vaccination does not enhance the protection

CD4(+)CD25(+) regulatory T cells (Tregs) do not only influence self-antigen specific immune responses, but also dampen the protective effect induced by a number of vaccines. The impact of CD4(+)CD25(+) Tregs on vaccines against schistosomiasis, a neglected tropical disease that is a major public health concern, however, has not been examined. In this study, a DNA vaccine encoding a 26 kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST) was constructed and its potential effects were evaluated by depleting CD25(+) cells prior to pVAX1-Sj26GST immunization. This work shows that removal of CD25(+) cells prior to immunization with the pVAX1-Sj26GST schistosomiasis DNA vaccine significantly increases the proliferation of splenocytes and IgG levels. However, CD25(+) cell-depleted mice immunized with pVAX1-Sj26GST show no improved protection against S. japonicum. Furthermore, depletion of CD25(+) cells causes an increase in both pro-inflammatory cytokines (e.g. IFN-γ, GM-CSF and IL-4) and an anti-inflammatory cytokine (e.g. IL-10), with CD4(+)CD25(-) T cells being one of the major sources of both IFN-γ and IL-10. These findings indicate that partial CD25(+) cell depletion fails to enhance the effectiveness of the schistosome vaccine, possibly due to IL-10 production by CD4(+)CD25(-) T cells, or other cell types, after CD25(+) cell depletion during vaccination.