CD4+CD25+Foxp3+ indirect alloreactive T cells from renal transplant patients suppress both the direct and indirect pathways of allorecognition

Alloreactive T cells recognize donor antigens by two routes: direct and indirect pathways of allorecognition. Although the direct pathway is reported to be dominant in allograft rejection, indirect allorecognition also plays an important role. Indirect alloreactivity is also observed in renal transplant patients irrespective of rejection. Previously we showed a predominance of interleukin (IL)-10 induced by indirect allorecognition of donor human leucocyte antigen (HLA)-DR peptides, suggesting the existence of indirect alloreactive T cells displaying regulatory activity. In the present work, our objective was to characterize these regulatory T cells. We detected indirect alloproliferation of peripheral blood mononuclear cells (PBMC) from renal transplant patients, induced by donor HLA-DR peptides, dependent on IL-4 or IL-10, suggesting regulatory activity as part of the alloreactive T-cell repertoire. PBMC-derived indirect alloreactive T-cell lines were established and produced both inflammatory and regulatory cytokines. We showed that two of these T-cell lines which were able to inhibit both direct and indirect alloproliferation of another T-cell line from the same patient presented a CD4(+)CD25(+)Foxp3(+) T-cell population. These data support the idea that indirect alloreactive T cells may also have regulatory activity and may contribute to the maintenance of the human renal allograft.     

Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ regulatory T cells

Naturally arising CD25(+)CD4(+) regulatory T (T(R)) cells can be exploited to establish immunologic tolerance to non-self antigens. In vivo exposure of CD25(+)CD4(+) T cells from normal naive mice to alloantigen in a T cell-deficient environment elicited spontaneous expansion of alloantigen-specific CD25(+)CD4(+) T(R) cells, which suppressed allograft rejection mediated by subsequently transferred naive T cells, leading to long-term graft tolerance. The expanded T(R) cells, which became CD25(low) in the absence of other T cells, stably sustained suppressive activity, maintained expression levels of other T(R) cell-associated molecules, including Foxp3, CTLA-4 and GITR, and could adoptively transfer tolerance to normal mice. Furthermore, specific removal of the T(R) cells derived from originally transferred CD25(+)CD4(+) T(R) cells evoked graft rejection in the long-term tolerant mice, indicating that any T(R) cells deriving from CD25(-)CD4(+) naive T cells minimally contribute to graft tolerance and that natural T(R) cells are unable to infectiously confer significant suppressive activity to other T cells. Similar antigen-specific expansion of T(R) cells can also be achieved in vitro by stimulating naturally present CD25(+)CD4(+) T cells with alloantigen in the presence of IL-2. The expanded CD25(+)CD4(+) T cells potently suppressed even secondary MLR in vitro and, by in vivo transfer, established antigen-specific long-term graft tolerance. Thus, in vivo or in vitro, direct or indirect ways of antigen-specific expansion of naturally arising Foxp3(+)CD25(+)CD4(+) T(R) cells can establish antigen-specific dominant tolerance to non-self antigens, and would also be instrumental in re-establishing self-tolerance in autoimmune disease and antigen-specific negative control of pathological immune responses.     

Direct in vivo evidence of CD4+ T cell requirement for CTL response and memory via pMHC-I targeting and CD40L signaling

CD4(+) T cell help contributes critically to DC-induced CD8(+) CTL immunity. However, precisely how these three cell populations interact and how CD4(+) T cell signals are delivered to CD8(+) T cells in vivo have been unclear. In this study, we developed a novel, two-step approach, wherein CD4(+) T cells and antigen-presenting DCs productively engaged one another in vivo in the absence of cognate CD8(+) T cells, after which, we selectively depleted the previously engaged CD4(+) T cells or DCs before allowing interactions of either population alone with na?ve CD8(+) T cells. This protocol thus allows us to clearly document the importance of CD4(+) T-licensed DCs and DC-primed CD4(+) T cells in CTL immunity. Here, we provide direct in vivo evidence that primed CD4(+) T cells or licensed DCs can stimulate CTL response and memory, independent of DC-CD4(+) T cell clusters. Our results suggest that primed CD4(+) T cells with acquired pMHC-I from DCs represent crucial "immune intermediates" for rapid induction of CTL responses and for functional memory via CD40L signaling. Importantly, intravital, two-photon microscopy elegantly provide unequivocal in vivo evidence for direct CD4-CD8(+) T cell interactions via pMHC-I engagement. This study corroborates the coexistence of direct and indirect mechanisms of T cell help for a CTL response in noninflammatory situations. These data suggest a new "dynamic model of three-cell interactions" for CTL immunity derived from stimulation by dissociated, licensed DCs, primed CD4(+) T cells, and DC-CD4(+) T cell clusters and may have significant implications for autoimmunity and vaccine design.     

A CFSE based assay for measuring CD4+CD25+ regulatory T cell mediated suppression of auto-antigen specific and polyclonal T cell responses

CD4(+)CD25(+) regulatory T cells (Tregs) are considered to play a key role as suppressors of immune mediated reactions. The analysis of Treg function in patients with autoimmune, allergic or oncogenic diseases has emerged over the past years. In the present study we describe a CFSE based protocol to measure Treg mediated suppression of CD4(+) T cells. Measuring Treg suppressive capacity towards proliferation of anti-CD3 Ab stimulated CD4(+)CD25(-) T cells in coculture experiments by means of a CFSE based and a classical [(3)H]thymidine incorporation assay gave similar results, provided that CD4(+)CD25(+) T cells were anergic. However, when CD4(+)CD25(+) T cells proliferated upon mitogenic stimulation, data obtained by the CFSE assay allowed the detection of a significant Treg suppression whereas this was clearly underestimated using the [(3)H]thymidine assay. In addition, an indirect CFSE based method was developed to analyze antigen specific responses of total CD4(+) T cells and Treg depleted CD4(+) T cells (i.e. CD4(+)CD25(-) T cells). Our results indicate that, in healthy individuals, CD4(+) T cell responses against the multiple sclerosis (MS) auto-antigens, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), were increased in Treg depleted CD4(+) T cells as compared to total CD4(+) T cells. Our initial data suggest that Tregs in MS patients show an impaired suppression of myelin reactive T cells when compared to healthy controls. Moreover, this experimental setup permits the measurement of cytokine production of the antigen proliferated CFSE(low) T cells by additional flow cytometric analyses. In conclusion, the described CFSE based Treg suppression assay is a valuable tool to study suppressor T cells in (auto)immune disorders.     

The direct pathway of human T-Cell allorecognition is not tolerized by stimulation with allogeneic peripheral blood mononuclear cells irradiated with high-dose ultraviolet B

Transfusions of high-dose (> or =10,000 Joule/m(2)) ultraviolet-B (UVB)-irradiated allogeneic leukocytes in rodent models have been shown to induce immunologic tolerance that is mediated by allospecific regulatory CD4(+) T cells. Whether these regulatory T cells recognize alloantigens through the direct or indirect pathway of allorecognition is controversial. Here, we demonstrate that the proliferative response obtained in standard primary mixed leukocyte reactions (MLRs) with human peripheral blood mononuclear cells (PBMCs) reflected a CD4(+) T-cell-dependent direct pathway of allorecognition and that high-dose UVB irradiation of PBMCs totally inhibited their capacity to induce a proliferative alloresponse. Re-stimulation with gamma-irradiated PBMCs from the same allogeneic donor (secondary MLR) elicited a proliferative and Th1-deviated response that was similar to the response induced in unprimed PBMCs. Finally, high-dose UVB was found to induce a rapid and massive apoptosis of irradiated PBMCs. Collectively, these data indicate that leukocytes irradiated with high-dose UVB are unable to prime for unresponsiveness or immune deviation in T cells directly recognizing allogeneic major histocompatibility complex molecules. Because it is well-established that antigens within transfused apoptotic cells are captured by resident tolerogenic spleen dendritic cells, we propose that tolerance induced by transfusions of high-dose UVB-irradiated leukocytes primarily involve T cells indirectly recognizing alloantigens.     

CD4+ T cells in adoptive immunotherapy and the indirect mechanism of tumor rejection

Tumor-specific CD4+ effector T cells often play a decisive role in immunologic tumor rejection, in some cases without evident co-participation of CD8+ T cells. During such CD4+ T-cell-mediated rejection there is often no detectable direct contact between T cells and tumor cells. Optimally prepared, adoptively transferred CD4+ T cells can reject established tumors with great efficiency even when targeted tumor cells express no MHC Class II molecules, implying that recognition of tumor antigen (Ag) occurs via MHC Class II-expressing host antigen-presenting cells (APC) within the tumor. Because consequent rejection also excludes Ag-specific contact between CD4+ T cells and MHC Class IIneg tumor cells, the most critical CD4+ T-cell-mediated event is likely cytokine release, resulting in an accumulation and activation of accessory cells such as tumoricidal macrophages and lymphokine-activated killer cells. Although such an indirect rejection mechanism may appear antithetical to popular strategies centered on CD8+ cytotoxic T cell (CTL), current evidence suggest that even CD8+ T-cell-mediated recognition/rejection often bypasses direct tumor cell contact and is largely cytokine mediated. While CTL are likely to participate prominently in many models of tumor rejection, indirect mechanisms of recognition/rejection have the theoretical advantage of remaining operative even when individual tumor cells evade direct contact by down-regulating MHC and/or Ag expression.     

Functional and phenotypic properties of peripheral T cells anergized by autologous CD3(+) depleted bone marrow cells

We have previously demonstrated that bone marrow cells (BMC) inhibit the generation of autologous Epstein-Barr virus (EBV) -specific cytotoxic T lymphocytes (CTL). It was also observed that CD3(+) cells obtained after 7 days of culture in the presence of autologous BMC could be used as inhibitors of EBV-CTL generation. In the present study, we examined these BMC induced regulator CD3(+) T cells with respect to phenotype, function, and T-cell activation pathways. We also questioned if the CD3(+) regulatory cell function is mediated by their direct effect on peripheral T cells or on the ability of antigen presenting cells (APC) to stimulate peripheral T cells. To answer this, CD3(+) cells from peripheral blood lymphocytes (PBL) were cultured with either CD3-depleted BMC or with CD3-depleted PBL. The CD3(+) cells were then isolated with immunomagnetic beads, designated as T(BM) and T(PBL), and were compared in functional studies. There was an increase in the expression of CD25 on T(BM) cells. The T(BM) cells also expressed less CD122 and a decreased number of CD3 molecules per cell. Both T(BM) and T(PBL) cell populations responded to mitogen (PHA) to the same magnitude. However, when stimulated through the CD3 complex with anti-CD3 monoclonal antibody (mAb), the T(BM) cells had a significantly decreased response than did T(PBL). The addition of IL-2 to these latter cultures augmented, but could not fully restore, the response. Additionally, stimulation of T(BM) cells with allogeneic cells failed to produce cytotoxic T cells. These "anergized" T(BM) and "nonanergized" (control) T(PBL) cells were added as third-party cells to a CTL generating culture of autologous PBL stimulated with allogeneic cells. The T(BM) cells exhibited suppressor function and inhibited the generation of CTL, in contrast with T(PBL). The effect of T(BM) cells on direct and indirect antigen presentation pathways demonstrated that T(BM) primarily effected indirect, but not direct, alloantigen presentation. To further explore the cytoplasmic T-cell activation events that occurred after the coculture of the PBL T cells with BMC, the levels of zeta-associated protein 70 (ZAP70) and extracellular receptor-activated kinase (ERK) were determined. There was a decrease in ZAP70 levels in the T(BM), which correlated with its reduced expression of cell surface CD3 and the attenuated response to anti-CD3 mAb activation. However, the activity of ERK was equally expressed by T(BM) and T(PBL). It, therefore, appears that the culturing of peripheral T cells with (non-T) BMC anergizes these cells (which become refractory to stimulation through the T-cell receptors), and induces immune suppressor function. These in vitro observations may provide a mechanism by which infused donor BMC serve to downregulate T-cell immunity.     

Human TSLP directly enhances expansion of CD8+ T cells

Human thymic stromal lymphopoietin (TSLP) promotes CD4(+) T-cell proliferation both directly and indirectly through dendritic cell (DC) activation. Although human TSLP-activated DCs induce CD8(+) T-cell proliferation, it is not clear whether TSLP acts directly on CD8(+) T cells. In this study, we show that human CD8(+) T cells activated by T-cell receptor stimulation expressed TSLP receptor (TSLPR), and that TSLP directly enhanced proliferation of activated CD8(+) T cells. Although non-stimulated human CD8(+) T cells from peripheral blood did not express TSLPR, CD8(+) T cells activated by anti-CD3 plus anti-CD28 did express TSLPR. After T-cell receptor stimulation, TSLP directly enhanced the expansion of activated CD8(+) T cells. Interestingly, using monocyte-derived DCs pulsed with a cytomegalovirus (CMV)-specific pp65 peptide, we found that although interleukin-2 allowed expansion of both CMV-specific and non-specific CD8(+) T cells, TSLP induced expansion of only CMV-specific CD8(+) T cells. These results suggest that human TSLP directly enhances expansion of CD8(+) T cells and that the direct and indirect action of TSLP on expansion of target antigen-specific CD8(+) T cells may be beneficial to adoptive cell transfer immunotherapy.     

Human anergic/suppressive CD4(+)CD25(+) T cells: a highly differentiated and apoptosis-prone population

Anergic/suppressive CD4(+)CD25(+) T cells exist in animal models but their presence has not yet been demonstrated in humans. We have identified and characterized a human CD4(+)CD25(+) T cell subset, which constitutes 7-10 % of CD4(+) T cells in peripheral blood and tonsil. These cells are a CD45RO(+)CD45RB(low) highly differentiated primed T cell population that is anergic to stimulation. Depletion of this small subset from CD4(+) T cells significantly enhances proliferation by threefold in the remaining CD4(+)CD25(-) T cells, while the addition of isolated CD4(+)CD25(+) T cells to CD4(+)CD25(-) T cells significantly inhibits proliferative activity. Blocking experiments suggest that suppression is not mediated via IL-4, IL-10 or TGF-beta and is cell-contact dependent. Isolated CD4(+)CD25(+) T cells are susceptible to apoptosis that is associated with low Bcl-2 expression, but this death can be prevented by IL-2 or fibroblast-secreted IFN-beta. However, the anergic/suppressive state of these cells is maintained after cytokine rescue. These human regulatory cells are therefore a naturally occurring, highly suppressive, apoptosis-prone population which are at a late stage of differentiation. Further studies into their role in normal and pathological situations in humans are clearly essential.     

Patients suffering from acute graft-versus-host disease after bone-marrow transplantation have functional CD4+CD25hiFoxp3+ regulatory T cells

Acute Graft-Versus-Host Disease (aGVHD), mediated by CD4(+) and CD8(+) effector T cells, is a life-threatening complication in hematopoietic stem cell (HSC) transplantation. Naturally-occurring CD4(+)CD25(hi)(Foxp3(+)) regulatory T cells (T(reg)) have been shown to modulate tolerance to aGVHD in murine graft models. In this report, we investigated their role in the prevention of aGVHD in patients transplanted with bone-marrow-derived HSC. When CD4(+)CD25(hi)Foxp3(+) T cells were isolated from bone-marrow grafts, they showed no suppressive activity. The analysis of their function in patients suffering from aGVHD after transplantation revealed a gain of suppressive activity indicating their inability to control the aGVHD induction. Thus, our findings clearly demonstrate that CD4(+)CD25(+) and CD4(+)CD25(hi)Foxp3(+) T cells, when administered in steady-state physiological conditions, do not influence the outcome of aGVHD after bone-marrow transplantation.     

Commentary: T cells get by with a little help from their friends

In animal models, lymphocytic choriomeningitis virus (LCMV) may be controlled after acute infection or may establish various levels of persistence. Cytotoxic responses mediated by CD8(+) T cells are responsible for both initial control of LCMV and for immunopathology. As discussed in this article, there is emerging evidence that the levels of antigen to which the immune system is exposed over time are important in controlling CD8(+) T cell activation, memory responses and exhaustion, and that these levels are affected by the efficiency of T cell help and the presence of antibody. To enable lasting control of LCMV infection, CD8(+) T cells, CD4(+) T cell help and B cells are all required. These findings have important implications for the prevention and treatment of infection by viruses such as hepatitis B and C viruses, cytomegalovirus and HIV. See accompanying article http://dx.doi.org/10.1002/eji.200324717     

Graft rejection mediated by CD4+ T cells via indirect recognition of alloantigen is associated with a dominant Th2 response

CD4(+) T cells that respond to indirectly presented alloantigen have been shown to mediate chronic rejection, however, the role of the indirect pathway in acute rejection has yet to be completely elucidated. To this end, BALB/c or C57BL/6 mice were depleted of CD8(+) T cells and transplanted with class II transactivator (CIITA)-deficient cardiac allografts, which cannot directly present class II alloantigens to CD4(+) T cells. In this manner, the rejection response by CD4(+) cells was forced to rely upon the indirect recognition pathway. When not depleted of CD8(+) cells, both BALB/c and C57BL/6 mice rejected CIITA-/- allografts and a polarized Th1 response was observed. In contrast, when BALB/c recipients of CIITA-/- allografts were depleted of CD8(+) T cells, the grafts were acutely rejected and a strong Th2 response characterized by eosinophil influx into the graft was observed. Interestingly, CD8-depleted C57BL/6 recipients of CIITA-/- allografts did not acutely reject their transplants and a Th2 response was not mounted. These findings indicate that CD4(+) T cells responding to indirectly presented alloantigens mediate graft rejection in a Th2-dominant manner, and provide further evidence for the role of Th2 responses in acute graft rejection.     

Role of bone marrow stromal cells in the generation of human CD8+ regulatory T cells

Fibroblast-like stromal cells exert a strong inhibitory effect on lymphocyte proliferation, both directly by interacting with responding lymphocytes and indirectly by inducing the generation of regulatory T cells. Indeed, upon triggering via the CD3/TCR complex, highly effective CD8(+)regulatory cells (CD8(+)Reg(c)) are generated from cocultures of peripheral blood CD8(+)T cells and bone-marrow-derived stromal cells. When cell-to-cell interactions occur, CD8(+)Reg(c) strongly inhibit lymphocyte proliferation at a ratio of 1:1 to 1:100 between CD8(+)Reg(c) and responding lymphocytes. Phenotypic analysis indicated that CD8(+)Reg(c) are CD25(+)CD28(+) and express low levels of mRNA for Foxp3 but they do not bear CTLA4 and glucocorticoid-induced tumor necrosis factor receptor antigens. Soluble mediators such as interleukin-10, transforming growth factor-beta, and prostaglandin E(2) are not involved in the generation of CD8(+)Reg(c) from CD8(+) precursors or in the immunosuppressive mechanism mediated by CD8(+)Reg(c) on lymphocyte proliferation. Cyclosporin A (CSA) slightly downregulated generation of CD8(+)Reg(c) indicating that only a small fraction of precursors of CD8(+)Reg(c) are sensitive to this immune-suppressive drug. Along this line, treatment of effector CD8(+)Reg(c)with CSA does not affect their immunosuppressive effect, indicating that the molecular mechanism of CD8(+)Reg(c)-mediated regulation is independent of the function of CSA biochemical target molecules.     

Dendritic cells crossprime allo-specific self-restricted CD4(+) T cells after coculture with dead allogeneic cells

Indirect alloreactivity, i.e., the recognition of allopeptides on self-MHC molecules, contributes both to acute and chronic rejection of transplants. The antigen presenting cell priming these allo-specific self-restricted T cells is unknown. We demontrate that dendritic cells, which have been matured in the presence of necrotic allogeneic cells, can crossprime allo-specific self-restricted CD4(+) T cells in vitro. We demonstrate dendrtitic cell mediated crosspriming of HLA-DR13 specific, HLA-DR7 restricted and HLA-DR1 specific, HLA-DR11 restricted CD4(+) T cells. The allo-specific self-restricted CD4(+) T cells primed in our culture system secrete predominantly Th1 and not Th2 cytokines. The use of dendritic cells to monitor the indirect pathway of alloreactivity should help to design and understand interventions against acute and chronic transplant rejection.     

Direct orthotopic transplantation of fresh surgical specimen preserves CD133+ tumor cells in clinically relevant mouse models of medulloblastoma and glioma

Recent identification of cancer stem cells in medulloblastoma (MB) and high-grade glioma has stimulated an urgent need for animal models that will not only replicate the biology of these tumors, but also preserve their cancer stem cell pool. We hypothesize that direct injection of fresh surgical specimen of MB and high-grade glioma tissues into anatomically equivalent locations in immune-deficient mouse brains will facilitate the formation of clinically accurate xenograft tumors by allowing brain tumor stem cells, together with their non-stem tumor and stromal cells, to grow in a microenvironment that is the closest to human brains. Eight of the 14 MBs (57.1%) and two of the three high-grade gliomas (66.7%) in this study developed transplantable (up to 12 passages) xenografts in mouse cerebellum and cerebrum, respectively. These xenografts are patient specific, replicating the histopathologic, immunophenotypic, invasive/metastatic, and major genetic (analyzed with 10K single nucleotide polymorphism array) abnormalities of the original tumors. The xenograft tumor cells have also been successfully cryopreserved for long-term preservation of tumorigenicity, ensuring a sustained supply of the animal models. More importantly, the CD133(+) tumor cells, ranging from 0.2%-10.4%, were preserved in all the xenograft models following repeated orthotopic subtransplantations in vivo. The isolated CD133(+) tumor cells formed neurospheres and displayed multi-lineage differentiation capabilities in vitro. In summary, our study demonstrates that direct orthotopic transplantation of fresh primary tumor cells is a powerful approach in developing novel clinical relevant animal models that can reliably preserve CD133(+) tumor cell pools even during serial in vivo subtransplantations. Disclosure of potential conflicts of interest is found at the end of this article.     

NK cells contribute to the skin graft rejection promoted by CD4+ T cells activated through the indirect allorecognition pathway

Rejection of solid organ allografts is promoted by T cells. Recipient T cells can directly recognize intact allo-MHC molecules on donor cells and can also indirectly recognize processed donor-derived allo-peptides presented by recipient antigen-presenting cells in the context of self-MHC molecules. Although CD4(+) T cells primed through the indirect allorecognition pathway alone are sufficient to promote acute allograft rejection, it is unknown how they can mediate graft destruction without cognate recognition of donor cells. In this study, we analyzed the indirect effector mechanism of skin allograft rejection using a mouse model in which SCID recipients bearing MHC class II-deficient skin allografts were adoptively transferred with CD4(+) T cells. Histologically, entire graft necrosis was preceded by mononuclear cell infiltration in the graft epithelia with epithelial cell apoptosis, indicating cell-mediated cytotoxicity against donor cells as an effector mechanism. Beside CD4(+) T cells and macrophages, NK cells infiltrated in the rejecting grafts. Depletion of NK cells as well as blocking of the activating NK receptor NKG2D allowed prolonged survival of the grafts. Expression of NKG2D ligands was up-regulated in the rejecting grafts. These results suggest that NK cells activated through NKG2D contribute to the skin allograft rejection promoted by indirectly primed CD4(+) T cells.     

Detection of low avidity CD8(+) T cell populations with coreceptor-enhanced peptide-major histocompatibility complex class I tetramers

The development of soluble recombinant peptide-major histocompatibility complex class I (pMHCI) molecules conjugated in multimeric form to fluorescent labels has enabled the physical quantification and characterization of antigen-specific CD8(+) T cell populations by flow cytometry. Several factors determine the binding threshold that enables visualization of cognate CD8(+) T cells with these reagents; these include the affinity of the T cell receptor (TCR) for pMHCI antigen. Here, we show that multimers constructed from peptide-human leukocyte antigen (pHLA) A0201 monomers engineered in the heavy chain alpha2 domain to enhance CD8 binding (K(D) approximately 85 microM) without impacting the TCR binding platform can detect cognate CD8(+) T cells bearing low affinity TCRs that are not visible with the corresponding wildtype pHLA A0201 multimeric complexes. Mechanistically, this effect is mediated by a disproportionate enhancement of the TCR/pMHCI association rate. In direct ex vivo applications, these coreceptor-enhanced multimers exhibit faithful cognate binding properties; concomitant increases in background staining within the non-cognate CD8(+) T cell population can be resolved phenotypically using polychromatic flow cytometry as a mixture of na?ve and memory cells. These findings provide the first validation of a novel approach to the physical detection of low avidity antigen-specific CD8(+) T cell populations; such coreceptor-enhanced multimeric reagents are likely to be useful in a multitude of settings for the detection of auto-immune, tumor-specific and cross-reactive CD8(+) T cells.