T cell subsets and in vitro immune regulation in "infectious" transplantation tolerance.

CD4-targeted mAb therapy results in permanent acceptance of cardiac allografts in rat recipients, in conjunction with features of the infectious tolerance pathway. Although CD4(+) T cells play a central role, the actual cellular and molecular tolerogenic mechanisms remain elusive. This study was designed to analyze in vitro alloimmune responses of T lymphocytes from CD4 mAb-treated engrafted hosts. Spleen, but not lymph node, cells lost proliferative response against donor alloantigen in MLR and suppressed test allograft rejection in adoptive transfer studies, suggesting compartmentalization of tolerogenic T cells in transplant recipients. A high dose of exogenous IL-2 restored the allogeneic response of tolerogenic T cells, indicating anergy as a putative mechanism. Vigorous proliferation of the tolerogenic T cells in in vivo MLR supports the existence of alloreactive lymphocytes in tolerogenic T cell repertoire and implies an active operational suppression mechanism. The tolerogenic splenocytes suppressed proliferation of naive splenocytes in vitro, consistent with their in vivo property of dominant immune regulation. Finally, CD45RC(+) but not CD45RC(-) T cells from tolerant hosts were hyporesponsive to alloantigen and suppressed the proliferation of normal T cells in the coculture assay. Thus, nondeletional, anergy-like regulatory mechanisms may operate via CD4(+)CD45RC(+) T cells in the infectious tolerance pathway in transplant recipients.     

CD11b (Mac-1): a marker for CD8+ cytotoxic T cell activation and memory in virus infection.

We have found that CD11b, a cell surface integrin of macrophages, granulocytes, and NK cells, is expressed by a subset of CD8+ T cells that include both the active virus-specific CTL and the virus-specific memory CTL populations. CD8+CD11b+ cells comprise less than 3% of naive mouse splenocytes, but after lymphocytic choriomeningitis virus (LCMV) infection increase by 9- to 12-fold by the peak (day 8) of the virus-specific CTL response. Depletion of day-8 splenocytes with anti-Mac-1 and C' or enrichment by sorting for CD11b+ or CD8+CD11b+ spleen cells demonstrated that LCMV-specific CTL are CD11b+. The CD11b+ subpopulation also contained the bulk of the IL-2-responsive CD8+ cells. MEL-14, a homing marker down-regulated on activated T cells, was down-regulated on the majority of CD8+ cells that became CD11b+. Less than 1% of LCMV-immune splenic lymphocytes expressed CD11b. Antibody and C' depletion of this population severely impaired the ability of immune splenocytes to respond to in vitro secondary stimulation with LCMV-infected peritoneal macrophages, but did not affect the generation of a primary allospecific CTL response in MLC. Mixing of CD8-depleted and CD11b-depleted LCMV-immune splenocytes failed to restore the ability of these cells to mount a virus-specific memory CTL response, indicating that a cell coexpressing CD8 and CD11b is essential for this response. As determined by limiting dilution analysis, the precursors for the LCMV-specific memory CTL response were enriched in the CD11b+ population of LCMV-immune splenocytes. CD11b stained far fewer CD8+ splenocytes from naive mice than did CD44 (Pgp-1), and among immune splenocytes it identified a small subpopulation of CD44hi cells, indicating that CD11b may be the best single marker available for discriminating between naive and memory CD8+ T cells.     

Liver sinusoidal endothelial cells tolerize T cells across MHC barriers in mice

Although livers transplanted across MHC barriers in mice are normally accepted without recipient immune suppression, the underlying mechanisms remain to be clarified. To identify the cell type that contributes to induction of such a tolerance state, we established a mixed hepatic constituent cell-lymphocyte reaction (MHLR) assay. Irradiated C57BL/6 (B6) or BALB/c mouse hepatic constituent cells (HCs) and CFSE-labeled B6 splenocytes were cocultured. In allogeneic MHLR, whole HCs did not promote T cell proliferation. When liver sinusoidal endothelial cells (LSECs) were depleted from HC stimulators, allogeneic MHLR resulted in marked proliferation of reactive CD4(+) and CD8(+) T cells. To test the tolerizing capacity of the LSECs toward alloreactive T cells, B6 splenocytes that had transmigrated through monolayers of B6, BALB/c, or SJL/j LSECs were restimulated with irradiated BALB/c splenocytes. Nonresponsiveness of T cells that had transmigrated through allogeneic BALB/c LSECs and marked proliferation of T cells transmigrated through syngeneic B6 or third-party SJL/j LSECs were observed after the restimulation. Transmigration across the Fas ligand-deficient BALB/c LSECs failed to render CD4(+) T cells tolerant. Thus, we demonstrate that Fas ligand expressed on naive LSECs can impart tolerogenic potential upon alloantigen recognition via the direct pathway. This presents a novel relevant mechanism of liver allograft tolerance. In conclusion, LSECs are capable of regulating a polyclonal population of T cells with direct allospecificity, and the Fas/Fas ligand pathway is involved in such LSEC-mediated T cell regulation.     

HIV replication leads to skewed maturation of CD8-positive T-cell responses in infected children

HIV-1 infection causes a severe T-cell impairment with alteration of immune response. However, in children the natural decline of lymphocytes and CD4 cells in early life makes it more difficult to monitor immunocompetence and progression of HIV-infection. Aim of this study was to characterize the CD8 response in non-vertically HIV-infected children exposed persistently to viremia and in HIV-infected children controlling efficiently viremia by ART, by analysing the effect of persistent viremia on CD4 and CD8 T-cells count, HIV-specific immune-response and naive/memory pattern of CD8 T-cell. Whereas, no differences of CD4 count between viremic patients and viral controllers were observed (1046.9 +/- 472.1 cells/microl vs 1101.3 +/- 415.4 cells/microl; p > 0.05), CD8 count was higher in the viremic patients (1080.6 +/- 652.1 cells/microl vs 747.5 +/- 389.9 cells/microl, p < 0.05). In viremic patients, HIV-specific CD8 T-cells correlated with viral load. However, in this group a loss of HIV-specific CD8 response was associated with a 7 fold decrease of na?ve and increase of pre-effector CD8 T-cells (62.8% +/- 10.21% vs 10.37% +/- 7.91%, p < 0.03). Persistent exposure to viremia alters HIV-specific CD8 response possibly through a persistent immune activation process leading to exhaustion of naive CD8 T-cells and skewed maturation of memory subset. Therefore, memory CD8 T-cells might lose the ability to respond correctly and efficiently to HIV-antigen exposure.     

Stimulator requirements for primed alloreactive T cells: macrophages and dendritic cells activate T cells across all genetic disparities

The cellular requirements for stimulating primed alloreactive T cells have been investigated. In vitro-primed secondary alloreactive cells, long-term lines, and Ly 1+2- noncytolytic clones which reacted with allo-H-2K, D, or Mls (M locus) antigens were tested. The data indicated that a specialized antigen-presenting cell such as a macrophage or a dendritic cell was required for stimulating primed alloreactive cells across all the genetic disparities tested. B and T lymphocytes were ineffective stimulators. The stimulator requirement for secondary and Ly 1+2- clone responses was heterogeneous, since both macrophages and dendritic cells were effective stimulators. Thus, the allostimulator requirement for inducing proliferation and mediator secretion by the primed T-cell populations closely paralleled the requirement for stimulating unprimed populations. The only exception found was the peritoneal washout population, which did not stimulate a primary response but did stimulate secondary responses. The failure of peritoneal macrophages to stimulate a primary response was shown to be due to an inhibitory pathway which did not occur when the responding population was alloantigen primed.     

Dual Role of miR-21 in CD4+ T-Cells: Activation-Induced miR-21 Supports Survival of Memory T-Cells and Regulates CCR7 Expression in Naive T-Cells

Immune cell-type specific miRNA expression patterns have been described but the detailed role of single miRNAs in the function of T-cells remains largely unknown. We investigated the role of miR-21 in the function of primary human CD4+ T-cells. MiR-21 is substantially expressed in T-cells with a memory phenotype, and is robustly upregulated upon αCD3/CD28 activation of both naive and memory T-cells. By inhibiting the endogenous miR-21 function in activated naive and memory T-cells, we showed that miR-21 regulates fundamentally different aspects of T-cell biology, depending on the differentiation status of the T-cell. Stable inhibition of miR-21 function in activated memory T-cells led to growth disadvantage and apoptosis, indicating that the survival of memory T-cells depends on miR-21 function. In contrast, stable inhibition of miR-21 function in activated naive T-cells did not result in growth disadvantage, but led to a significant induction of CCR7 protein expression. Direct interaction between CCR7 and miR-21 was confirmed in a dual luciferase reporter assay. Our data provide evidence for a dual role of miR-21 in CD4+ T cells; Regulation of T-cell survival is confined to activated memory T-cells, while modulation of potential homing properties, through downregulation of CCR7 protein expression, is observed in activated naive T-cells.     

T-cell proliferation in vivo and the role of cytokines

Unlike typical naive T cells, T cells with an activated (CD44hi) memory phenotype show a rapid rate of proliferation in vivo. The turnover of memory-phenotype CD8+ T cells can be considerably augmented by injecting mice with various compounds, including polyinosinic polycytidylic acid, lipopolysaccharide and immunostimulatory DNA (CpG DNA). Certain cytokines, notably type I (alpha, beta) interferons (IFN-I), have a similar effect. These agents appear to induce proliferation of CD44hi CD8+ cells in vivo by an indirect process involving production of effector cytokines, possibly interleukin-15, by antigen-presenting cells. Although none of the agents tested induces proliferation of naive-phenotype T cells, IFN-I has the capacity to cause upregulation of surface markers on purified naive T cells. Depending upon the experimental conditions used, IFN-I can either inhibit or enhance primary responses of naive T cells to specific antigen.     

Intrinsic differences in the proliferation of naive and memory human B cells as a mechanism for enhanced secondary immune responses

Humoral immune responses elicited after secondary exposure to immunizing Ag are characterized by robust and elevated reactivity of memory B cells that exceed those of naive B cells during the primary response. The mechanism underlying this difference in responsiveness of naive vs memory B cells remains unclear. We have quantitated the response of naive and memory human B cells after in vitro stimulation with T cell-derived stimuli. In response to stimulation with CD40 ligand alone or with IL-10, both IgM-expressing and Ig isotype-switched memory B cells entered their first division 20-30 h earlier than did naive B cells. In contrast, the time spent traversing subsequent divisions was similar. Consistent with previous studies, only memory cells differentiated to CD38(+) blasts in a manner that increased with consecutive division number. These differentiated CD38(+) B cells divided faster than did CD38(-) memory B cell blasts. Proliferation of CD40 ligand-stimulated naive B cells as well as both CD38(+) and CD38(-) cells present in cultures of memory B cells was increased by IL-10. In contrast, IL-2 enhanced proliferation of CD38(-) and CD38(+) memory B cell blasts, but not naive cells. Thus, memory B cells possess an intrinsic advantage over naive B cells in both the time to initiate a response and in the division-based rate of effector cell development. These differences help explain the accelerated Ab response exhibited by memory B cells after secondary challenge by an invading pathogen, a hallmark of immunological memory.     

Flt3 ligand-generated murine plasmacytoid and conventional dendritic cells differ in their capacity to prime naive CD8 T cells and to generate memory cells in vivo

Mature dendritic cells (DCs) have the capacity to induce efficient primary T cell response and effector cell differentiation. Thus, these cells are a major tool in the design of various immunotherapeutic protocols. We have tested the capacity of different subsets of matured DCs pulsed with a peptide to induce the differentiation of naive CD8 T cells into memory cells in vivo. Flt3 ligand (FL) induces the differentiation of conventional DCs (cDCs) and plasmacytoid DCs (PDCs) from murine bone marrow precursors in vitro. After maturation, both subsets become strong stimulators of Ag-specific T cell responses in vitro. However, the in vivo T cell stimulatory capacity of these DC subsets has not been studied in detail. In the present study, we demonstrate that mature FL-generated DCs induce efficient peptide-specific CD8 T cell response and memory cell differentiation in vivo. This is mainly due to the cDC subset because the PDC subset induced only a negligible primary CD8 response without detectable levels of memory CD8 T cell differentiation. Thus, in vitro FL-generated mature cDCs, but not PDCs, are potent stimulators of peptide-specific CD8 T cell responses and memory generation in vivo.     

Effector and memory CD8+ T cells can be generated in response to alloantigen independently of CD4+ T cell help

There is now considerable evidence suggesting that CD8(+) T cells are able to generate effector but not functional memory T cells following pathogenic infections in the absence of CD4(+) T cells. We show that following transplantation of allogeneic skin, in the absence of CD4(+) T cells, CD8(+) T cells become activated, proliferate, and expand exclusively in the draining lymph nodes and are able to infiltrate and reject skin allografts. CD44(+)CD8(+) T cells isolated 100 days after transplantation rapidly produce IFN-gamma following restimulation with alloantigen in vitro. In vivo CD44(+)CD8(+) T cells rejected donor-type skin allografts more rapidly than naive CD8(+) T cells demonstrating the ability of these putative memory T cells to mount an effective recall response in vivo. These data form the first direct demonstration that CD8(+) T cells are able to generate memory as well as effector cells in response to alloantigen during rejection in the complete absence of CD4(+) T cells. These data have important implications for the design of therapies to combat rejection and serve to reinforce the view that CD8(+) T cell responses to allografts require manipulation in addition to CD4(+) T cell responses to completely prevent the rejection of foreign organ transplants.     

CD40 stimulation of human peripheral B lymphocytes: distinct response from naive and memory cells

During secondary immune response, memory B lymphocytes proliferate and differentiate into Ig-secreting cells. In mice, the binding of CD40 by CD154 clearly enhances the activation and differentiation of memory B lymphocytes. In humans, the role of CD40-CD154 in the stimulation of memory B lymphocytes is not as obvious since in vitro studies reported positive and negative effects on their proliferation and differentiation in Ig-secreting cells. In this study, we examine the response of peripheral memory and naive cells in relation to the duration of CD40-CD154 interaction. We measured the proliferation and differentiation of both subsets stimulated with CD154 and IL-4 for short- (4-5 days) and long-term (>7 days) periods. Following short-term stimulation, memory B lymphocytes did not expand but represented the only subset differentiating into IgG- and IgM-secreting cells. A longer stimulation of this population led to cell death, while promoting naive B lymphocyte proliferation, expansion, and differentiation into IgM- or IgG-secreting cells. This prolonged CD40 stimulation also triggered naive B lymphocytes to switch to IgG and to express CD27 even in absence of somatic hypermutation, suggesting that these latter events could be independent. This study suggests that naive and memory B lymphocytes have distinct requirements to engage an immune response, reflecting their different roles in humoral immunity.     

Suppression of cytotoxic and proliferative xenogeneic T-cell responses by transgenic expression of indoleamine 2,3-dioxygenase

Tryptophan catabolism initiated by the enzyme indoleamine 2,3-dioxygenase (IDO) has been postulated to be a natural regulatory mechanism for T cells. In this study, we generated a pig endothelial cell line expressing full-length human IDO (P-HuIDO) to serve as a simple model of a cellular xenogeneic graft. Splenocytes from mice primed to P-HuIDO cells were found to be as responsive to secondary stimulation as splenocytes from mice primed to parental cells. However, in T-cell proliferation assays using P-HuIDO cells as stimulators, a significant inhibition of both naive and memory xenogeneic proliferative responses was noted. Furthermore, the production of interferon-gamma and cytotoxic T lymphocyte function were also affected. When severe combined immunodeficiency mice were grafted with P-HuIDO cells, then challenged with primed splenocytes from BALB/c mice, cellular infiltration to the graft was delayed. Our findings suggest that transgenic expression of IDO in xenografts contributes to prolonged graft survival.     

Endogenous myelin basic protein is presented in the periphery by both dendritic cells and resting B cells with different functional consequences

Multiple sclerosis is an inflammatory disease believed to be triggered by erroneous activation of self-reactive T cells specific for myelin proteins such as myelin basic protein (MBP). Inflammation is limited to the CNS, suggesting that the myelin-specific T cells encounter their Ags only after they cross the blood-brain barrier. However, our previous studies in mice showed that MBP epitopes are constitutively presented in lymphoid tissues. Here we identified which APCs in lymph nodes present endogenous MBP epitopes and determined the functional consequences of this presentation for both naive and activated MBP-specific T cells. Both CD8alpha+ and CD8alpha- dendritic cells were potent stimulators of proliferation for both naive and previously activated/memory MBP-specific T cells. Surprisingly, resting B cells also presented endogenous MBP that was acquired using a BCR-independent mechanism. Interaction with resting B cells triggered proliferation of both naive and activated MBP-specific T cells. Activated/memory MBP-specific T cells proliferating in response to resting B cells presenting endogenous MBP did not produce cytokines and became more refractory to subsequent stimulation. Interestingly, cytokine production by activated/memory T cells was triggered by resting B cells if the number of MBP epitopes presented was increased by adding exogenous MBP peptide. These results suggest that activated MBP-specific T cells may become less pathogenic in vivo following encounter with resting B cells presenting steady-state levels of endogenous MBP but can expand and remain pathogenic if the amount of MBP presented by B cells is increased, which could occur during chronic demyelinating disease.     

Defective CD8+ T cell activation and cytolytic function in the absence of LFA-1 cannot be restored by increased TCR signaling.

Signaling through the TCR as well as engagement of costimulatory molecules are required for efficient T cell activation and progression into differentiated effector cells. The beta2 integrin LFA-1 (CD11a/CD18) has been implicated in TCR costimulation as well as in cell-cell adhesion function, but its exact role is still ambiguous. The present study focuses on the requirement for LFA-1 in CD8+ T cell activation and effector function using LFA-1-deficient cells expressing the 2C transgenic TCR as a model system. The lack of LFA-1 expression in 2C T cells resulted in severely diminished proliferative response toward allogeneic BALB/c splenocytes. Increase in TCR signaling alone by pulsing stimulators with high affinity peptides, p2Ca or QL9, had minimal effects in restoring proliferation. Addition of exogenous IL-2, however, enhanced the effect of peptide pulsing on proliferation of LFA-1-deficient 2C T cells. LFA-1-deficient 2C CTLs generated from alloantigen stimulation exhibited a defective cytotoxic activity when tested on a variety of target cells. Cytolysis could be improved, but not fully rectified by peptide pulsing of target cells. Thus, in the 2C TCR model, LFA-1 has a requisite role for optimal CD8+ T cell activation and effector function, which cannot be overcome by increasing peptide/MHC density on either the APCs or target cells, respectively.     

The heat shock proteins,Hsp70 and Hsp83, of Leishmania infantum are mitogens for mouse B cells

Extending earlier studies, this report demonstrates that Leishmania infantum heat shock proteins (Hsps), Hsp70 and Hsp83, expressed as recombinant proteins fused to the Escherichia coil maltose-binding protein (MBP), are potent mitogens for murine splenocytes. The response was not due to lipopolysaccharide (LPS) because the stimulatory activity of Hsp preparations was sensitive to boiling and trypsin treatments, whereas the corresponding activity of LPS was resistant to both treatments. It was found that in vitro incubation of spleen cells with the Leishmania Hsps leads to the expansion of CD220-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. In fact, splenocytes from B cell-deficient mice did not proliferate in response to the Leishmania Hsps. In contrast, spleen cells from athymic nude mice were significantly stimulated by these recombinant proteins as an indication that the MBP-Hsp70 and MBP-Hsp83 recombinant proteins behave as T cell-independent mitogens of B cells. Furthermore, both proteins were able to induce proliferation on B cell populations purified from BALB/c spleen.     

Effect of mitogenic stimulation and heat shock on protein syntheses in human T cells

Effects of moderate (42 degrees C, 1 hour) and strong (44 degrees C, 1 hour) heat shocks on resting (TR) and phytohemagglutinin stimulated human T-cells (TP) were studied. Both treatments were shown to cause in the latter considerable fall of the level of protein synthesis, as compared to resting cells. Mitogen-stimulated cells stopped their proliferation irreversibly and part of them (approx: 40%) died after even mild shock (at 42 degrees C). Following heat treatment in both the cell types the synthesis of heat shock 70 and 90 kDa proteins was induced which was much more pronounced in TR. These and earlier results of the authors allow a conclusion that involvement of cells in active proliferation may decrease their resistance to stress, and that this phenomenon coincides with the diminishing in synthesis and accumulation of stress proteins.     

Enhanced expression of heat shock protein and mRNA synthesis by type I interferon in human HL-60 leukemic cells

The effects of IFN and mild hyperthermia on the responses of human promyelocytic HL-60 cells were investigated. Cells subjected to an elevated culture temperature (39.5 degrees-40.5 degrees C instead of 37 degrees C, herein referred to as heat-treated cells) showed an increase in heat shock proteins (HSPs) and corresponding mRNA synthesis, which were additionally potentiated by the presence of IFN. With cells cultured at 37 degrees C, IFN had no effect on HSP expression. The observed inhibition (40-70%) of RNA polymerase II-directed RNA synthesis (based on alpha-amanitin sensitivity) in isolated nuclei of heat-treated cells was also significantly reversed by the simultaneous addition of IFN. These data suggest that the IFN-amplified HSP gene expression may be involved in preventing irreversible damage or in fine tuning the recovery of mammalian cells from heat stress.     

Expression of the IL-7 Receptor Alpha-Chain Is Down Regulated on the Surface of CD4 T-Cells by the HIV-1 Tat Protein

HIV infection elicits defects in CD4 T-cell homeostasis in both a quantitative and qualitative manner. Interleukin-7 (IL-7) is essential to T-cell homeostasis and several groups have shown reduced levels of the IL-7 receptor alpha-chain (CD127) on both CD4 and CD8 T-cells in viremic HIV+ patients. We have shown previously that soluble HIV Tat protein specifically down regulates cell surface expression of CD127 on human CD8 T-cells in a paracrine fashion. The effects of Tat on CD127 expression in CD4 T-cells has yet to be described. To explore this effect, CD4 T-cells were isolated from healthy individuals and expression levels of CD127 were examined on cells incubated in media alone or treated with Tat protein. We show here that, similar to CD8 T-cells, the HIV-1 Tat protein specifically down regulates CD127 on primary human CD4 T-cells and directs the receptor to the proteasome for degradation. Down regulation of CD127 in response to Tat was seen on both memory and naive CD4 T-cell subsets and was blocked using either heparin or anti-Tat antibodies. Tat did not induce apoptosis in cultured primary CD4 T-cells over 72 hours as determined by Annexin V and PI staining. Pre-incubation of CD4 T-cells with HIV-1 Tat protein did however reduce the ability of IL-7 to up regulate Bcl-2 expression. Similar to exogenous Tat, endogenously expressed HIV Tat protein also suppressed CD127 expression on primary CD4 T-cells. In view of the important role IL-7 plays in lymphocyte proliferation, homeostasis and survival, down regulation of CD127 by Tat likely plays a central role in immune dysregulation and CD4 T-cell decline. Understanding this effect could lead to new approaches to mitigate the CD4 T-cell loss evident in HIV infection.     

Roles of I-E molecule and CD28 costimulation in induction of suppression by staphylococcal enterotoxin B in vivo.

Exposure to bacterial superantigens leads to the induction of a subsequent state of immune hyporesponsiveness. Using a transwell coculture system, a previous report demonstrated that splenocytes from staphylococcal enterotoxin B (SEB)-injected BALB/c mice secreted soluble mediators to suppress the proliferative response of naive syngeneic splenocytes to SEB stimulation. We show in the present study that, in contrast to the suppressive effect induced by SEB in BALB/c (H-2(d) haplotype), MRL(+/+), and MRL-lpr/lpr (H-2(k)) mice, SEB-primed splenocytes from I-E(-) strains such as B6, B10, A.BY (H-2(b)), and A.SW (H-2(s)) mice failed to inhibit the CD25 expression and the proliferative activity of their syngeneic naive responder splenocytes. Further results revealed that the SEB-primed cells from BALB/c, but not B6, mice inhibited the CD25 expression and proliferation of naive responder cells from either BALB/c or B6 mice, indicating the critical regulatory role of the effector cells. Unlike SEB, staphylococcal enterotoxin A induced profound suppression in both BALB/c and B6 mice. Moreover, the suppressive competence of SEB-primed splenocytes was diminished in CD28-deficient BALB/c mice. Taken together, our results indicate that when SEB is used as a stimulator in vivo, both the I-E molecule and CD28 costimulation are required for the induction of regulatory cells bearing suppressive activity.