Host APCs Research Articles

Intervention with costimulatory pathways as a therapeutic approach for graft-versus-host disease

Graft-versus-host disease (GVHD) is mediated by mature donor T cells contained in the hematopoietic stem cell graft. During the development of GVHD, signaling through a variety of costimulatory receptors plays an important role in allogeneic T cell responses. Even though delivery of costimulatory signals is a prerequisite for full activation of donor T cells in the phase of their interactions with host APCs, their involvement with GVHD might occur over multiple stages. Like many other aspects of GVHD, promise of therapeutic interventions with costimulatory pathways has been gleaned from preclinical models. In this review, I summarize some of the advances in roles of costimulatory molecules in GVHD pathophysiology and discuss preclinical approaches that warrant further exploration in the clinic, focusing on novel strategies to delete pathogenic T cells.

Extracorporeal photopheresis for the prevention of acute GVHD in patients undergoing standard myeloablative conditioning and allogeneic hematopoietic stem cell transplantation

GVHD is partly mediated by host APCs that activate donor T cells. Extracorporeal photopheresis (ECP) can modulate APC function and benefit some patients with GVHD. We report the results of a study using ECP administered before a standard myeloablative preparative regimen intended to prevent GVHD. Grades II-IV acute GVHD developed in 9 (30%) of 30 recipients of HLA-matched related transplants and 13 (41%) of 32 recipients of HLA-matched unrelated or HLA-mismatched related donor transplants. Actuarial estimates of overall survival (OS) at day 100 and 1-year post transplant were 89% (95% CI, 78-94%) and 77% (95% CI, 64-86%), respectively. There were no unexpected adverse effects of ECP. Historical controls receiving similar conditioning and GVHD prophylaxis regimens but no ECP were identified from the database of the Center for International Blood and Marrow Transplant Research and multivariate analysis indicated a lower risk of grades II-IV acute GVHD in patients receiving ECP (P=0.04). Adjusted OS at 1 year was 83% in the ECP study group and 67% in the historical control group (relative risk 0.44; 95% CI, 0.24-0.80) (P=0.007). These preliminary data may indicate a potential survival advantage with ECP for transplant recipients undergoing standard myeloablative hematopoietic cell transplantation.

Expression of constitutively-active aryl hydrocarbon receptor in T-cells enhances the down-regulation of CD62L, but does not alter expression of CD25 or suppress the allogeneic CTL response

Activation of aryl hydrocarbon receptor (AhR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in T-cells is required for TCDD-induced suppression of the allogeneic CTL response and for induction of CD25hiCD62Llow adaptive regulatory T-cells. Here, the ability of a constitutively-active AhR (CA-AhR) expressed in T-cells alone to replicate the effects of TCDD was examined. The response of CA-AhR-expressing B6 donor T-cells in B6xD2F1 mice was compared to the response of wild-type B6 donor T-cells in B6xD2F1 mice given a single dose of TCDD. Expression of CA-AhR in donor T-cells enhanced the down-regulation of CD62L on Day 2 after injection, similar to a single oral dose of TCDD, but did not induce up-regulation of CD25 on Day 2 or affect CTL activity on Day 10. This suggests that activation of AhR in T-cells alone may not be sufficient to alter T-cell responses in this acute graft-versus-host (GvH) model. Since host APC are responsible for activating the donor T-cells, we examined the influence of the F1 host’s AhR on donor T-cell responses by creating an AhR−/− B6xD2F1 host that had a greatly diminished AhR response to TCDD compared to wild-type F1 mice. As in AhR+/+ B6xD2F1 mice, the CTL response in AhR−/− B6xD2F1 mice was completely suppressed by TCDD. This suggests that either CA-AhR dose not fully replicate the function of TCDD-activated AhR in suppression of the CTL response, or that minimal activation of AhR in host cells is required to combine with activation of AhR in T-cells to elicit the immunosuppressive effects of TCDD.

TLR Agonists Prevent the Establishment of Allogeneic Hematopoietic Chimerism in Mice Treated with Costimulation Blockade

Activation of TLR4 by administration of LPS shortens the survival of skin allografts in mice treated with costimulation blockade through a CD8 T cell-dependent, MyD88-dependent, and type I IFN receptor-dependent pathway. The effect of TLR activation on the establishment of allogeneic hematopoietic chimerism in mice treated with costimulation blockade is not known. Using a costimulation blockade protocol based on a donor-specific transfusion (DST) and a short course of anti-CD154 mAb, we show that LPS administration at the time of DST matures host alloantigen-presenting dendritic cells, prevents the establishment of mixed allogeneic hematopoietic chimerism, and shortens survival of donor-specific skin allografts. LPS mediates its effects via a mechanism that involves both CD4(+) and CD8(+) T cells and results from signaling through either the MyD88 or the type I IFN receptor pathways. We also document that timing of LPS administration is critical, as injection of LPS 24 h before treatment with DST and anti-CD154 mAb does not prevent hematopoietic engraftment but administration the day after bone marrow transplantation does. We conclude that TLR4 activation prevents the induction of mixed allogeneic hematopoietic chimerism through type I IFN receptor and MyD88-dependent signaling, which leads to the up-regulation of costimulatory molecules on host APCs and the generation of alloreactive T cells. These data suggest that distinct but overlapping cellular and molecular mechanisms control the ability of TLR agonists to block tolerance induction to hematopoietic and skin allografts in mice treated with costimulation blockade.

Recruitment of Donor T Cells to Secondary Lymphoid Tissue Is Enhanced by Conditioning Therapy and Donor T Cells/Marrow

Background: Previous investigators have demonstrated that secondary lymphoid tissue (SLT) including the spleen is critical for the initial activation of donor T cells after allogeneic stem cell transplantation (SCT) leading to GVHD. This work showed significant redundancy in the role of all SLT including the spleen and suggested that approaches to prevent GVHD would need to inhibit the migration of donor T cells to all SLT. The mechanisms mediating the migration of donor T-cells to SLT have not been well characterized and the specific role of conditioning therapy in this process has not been investigated. Thus, here we sought to evaluate the expression of trafficking proteins by host antigen presenting cells isolated from the MLN and spleen.Methods: B6D2 recipient mice that underwent conditioning were lethally irradiated the night prior to transplantation. The following day recipient mice were transplanted i.v. with 3 × 106 T cell depleted bone marrow cells with 5 × 106 column purified T-cells. T-cells were from eGFP C57Bl/6 transgenic mice. Six hours after transplantation, recipient mice were sacrificed and the MLN and spleen removed. Dendritic cells (DCs) were isolated by collagenase digestion and mechanical disruption and sorted on a high speed MoFlow sorter using anti-CD11c monoclonal antibodies. Gates were drawn around CD11c high cells that did not express eGFP. 3–5 × 104 DCs from the MLN and 1–2 × 105 DCs from the spleen were pooled from five different mice per group, lysed in an RNA amplification buffer and analyzed either by real-time PCR or Agilent Whole Mouse Genome Microarray Chips.Results: Four different groups of mice were analyzed including one group that received no treatment, one that received irradiation alone, one that received irradiation plus allogeneic bone marrow and T cells, and one that received allogeneic bone marrow and T cells without irradiation. By comparing recipients that received allogeneic bone marrow and T cells, with those that received conditioning therapy, allogeneic bone marrow and T cells, we found that conditioning therapy significantly induced the expression by host DCs of the chemokines, CXCL10, CCL17, CCL20 and CCL22 from DCs isolated from both the MLN and the spleen. CCL3 expression by host cells was only upregulated from DCs isolated from the MLN and not the spleen. Conditioning therapy downregulated the expression of CCL1, CCL2 and CCL4 from DCs isolated from both the MLN and spleen (Table 1). Conditioning therapy markedly enhanced the expression by splenic and MLN DCs of STAT4 and p40 IL-12.Table 1MLNSpleenCXCL1014.820CCL310.9−1.0CCL177.05.1CCL222.47.8CCL202.21.6CCL61.31.2CCL5−1.33.0CCL19−1.4−2.3CXCL9−1.5−1.3CCL1−2.22.2CCL2−3.7−3.0CCL4−6.0−1.4Next we analyzed the effect of allogeneic bone marrow and T cells on the expression of chemokine ligands and signaling proteins by splenic and MLN DCs. Interestingly, the receipt of allogeneic bone marrow plus T cells and conditioning therapy led to a marked increase in the expression of chemokine ligands by DCs isolated from the spleen with little effect on the expression of chemokine ligands by DCs isolated from the MLN compared to mice that only received irradiation but did not undergo allogeneic transplantationTable 2.MLNSpleenCXCL91.157CCL222.236CCL5−1.134CXCL102.130CCL174.38.1CCL4−1.17.2CCL61.46.7CCL36.01.0CCL202.41.0CCL12.5−1.5CCL19−4.2−4.0CCL2−6.7−7.1The receipt of irradiation, donor bone marrow and T cells markedly enhanced the expression by host DCs from the spleen of STAT4 and p40 IL-12 while downregulating the expression of STAT6.We confirmed that the expression of CXCL9, CXCL10, CCL4 and CCL5 were markedly increased by host APCs using real time PCR. Finally, we demonstrated in vitro that donor T cell production of IFN-g was critical to the generation of CXCL9 and CXCL10 by host APCs.Conclusion: Conditioning therapy markedly enhances the expression of chemokine ligands and proteins important in Th1 T cell activation by host APCs. However, the expression was enhanced the most in splenic DCs isolated from recipient mice that had received irradiation with allogeneic bone marrow and allogeneic T cells. Our work suggests that the processes mediating the migration of donor T cells to the spleen may differ from those in the MLN and that the CXCR3 binding chemokines, CXCL9 and CXCL10 along with the CCR5 binding chemokine, CCL3, may be quite critical for donor T cell homing to the spleen.

Leukemic Blasts Acting as Host Antigen Presenting Cells Trigger a Combined CD4 and CD8 Allo-Immune Response Directed against Mismatched HLA Class I

HLA disparity between patient and donor increases the risk and the severity of graft versus host disease (GVHD). In order to safely administer HLA mismatched stem cell transplantation (SCT) and donor lymphocyte infusion (DLI), it is important to understand the mechanisms underlying GVHD in HLA mismatched settings. The degree of patient chimerism, especially of the antigen presenting cells, at the time of DLI has been shown to influence the development of GVHD. This is suggested by the correlation between the time after SCT of DLI administration and the occurrence of GVHD. In mouse models the underlying role of the presence of host APCs in the occurrence of GVHD has been demonstrated in both MHC matched and mismatched models. In this study, the alloimmune response in a patient suffering from GVHD after an HLA class I mismatched DLI was characterized. The patient received a T-cell depleted SCT followed by two DLIs from the same donor. The SCT and first DLI, administered for mixed chimerism, did not result in any signs of GVHD. In contrast, the second DLI led to severe GVHD. Patient chimerism in different cell subsets present prior to the two DLIs showed no significant difference in patient T-cell chimerism and there were no patient B-cell, monocytes or dendritic cells present prior to each DLI. However, a significant difference in the presence of patient leukemic blasts was found at the time of the two DLIs. Prior to the first DLI no leukemic cells could be demonstrated, whereas 9% leukemic blasts were present in bone marrow prior to the second DLI. We showed that the leukemic blasts expressed HLA class II. Correlating with the absence of GVHD, the first DLI did not lead to activation of donor T-cells while the second DLI led to severe GVHD accompanied by a vigorous CD8 and CD4 T-cell response. Activated donor derived CD8 and CD4 T-cells from this response were isolated by single cell sort based on HLA-DR expression. Expanded clones were characterized for alloreactivity, HLA restriction, specificity and clonality. We showed that 50 of the 53 expanded CD8 T-cells were alloreactive and that all alloreactive CD8 T-cells were restricted to the mismatched HLA-A2. Additionally, by sequencing the T-cell receptor (TCR) Vβ CDR3 region, we showed that all alloreactive CD8 clones expressed a different CDR3 region, illustrating polyclonality. Of the 88 expanded CD4 clones, 21 were alloreactive and the majority of the alloreactive CD4 T-cell clones were directed against an HLA-A2 derived peptide presented in HLA-DR1. By Vβ analysis we showed that the CD4 T-cell response was also polyclonal. Finally, to investigate the role of the leukemic blasts in the initiation of the GVHD, the capacity of the different cell subsets present prior to DLI 1 or 2 to stimulate donor CD8 and CD4 T-cells was analyzed. This showed that only the leukemic blasts were able to highly stimulate both the CD8 and the CD4 T-cells. Our data indicate that the leukemic blasts by acting as host APCs triggered a combined CD4 and CD8 allo-immune response directed against the mismatched HLA, and thus initiated not only anti-leukemic reactivity but also lethal GVHD.

Induction of Invariant NKT Cell-Dependent Alloreactivity by Administration of G-CSF after Bone Marrow Transplantation

G-CSF is often used to hasten neutrophil recovery following allogeneic bone marrow transplantation (BMT), but the clinical and immunological consequences invoked remain unclear. We examined this in murine models and found that administration of both standard G-CSF and pegylated G-CSF early after BMT significantly increased graft-versus-host disease (GVHD). This effect was seen in the B6 → B6D2F1, BALB/c → B6 and C3H.SW → B6 systems of GVHD to either MHC or multiple minor histocompatibility antigens. This effect was dependent on total body irradiation (TBI) rendering host dendritic cells (DC) responsive to G-CSF by up-regulating their expression of the G-CSF receptor as determined by real-time PCR. This induction of G-CSFR expression was not seen following busulfan (Bu), cyclophosphamide (Cy) or fludarabine. The enhanced GVHD was present when G-CSF was administered to both WT and G-CSFR−/− donors but not G-CSFR−/− recipients, confirming that host signalling was critical for this effect. G-CSF administration after BMT had no effect on inflammatory cytokine generation but enhanced in vivo CTL activity after BMT when administered to WT but not G-CSFR−/−, CD1d−/−, IFNgR−/− or CD40−/− recipients. Furthermore, donor iNKT cell activation was absent in CD11c Diptheria Toxin Receptor recipient transgenic mice depleted of dendritic cells (DC) by diphtheria toxin and treated with G-CSF. Thus, stimulation of host DC by G-CSF subsequently unleashed a cascade of events characterized by CD1d dependent donor iNKT cell activation, IFNg secretion and CD40-dependent amplification of donor CTL function during the effector phase of GVHD. Critically, the detrimental effects of G-CSF on GVHD were present when administered early following TBI conditioning and at a time when residual host APC were still present (day +1), but had no effect when administered at day +8 when host DC were not detectable by phenotypic or functional analysis. This is consistent with the inefficient cross presentation of host Ag within MHC class I by donor DC after BMT. In addition, the administration of G-CSF after Bu/Cy conditioning had no effect, perhaps explaining the conflicting and somewhat controversial clinical studies from the large European and North American BMT registries since TBI conditioning predominated only in the positive European study. These data have major implications for the use of G-CSF in disease states where NKT cell activation may have important effects on outcome and suggest a guide to the safe use of G-CSF after allogeneic BMT.

Cross Presentation of Antigen on MHC Class II via the Draining Lymph Node after Corneal Transplantation in Mice

We investigated Ag trafficking from the cornea and T effector cell activation in secondary lymphoid tissue after corneal transplantation. In preliminary experiments, the central cornea was shown to contain a population of CD45(+), CD11b(+), CD11c- cells, with a few MHC class II(+) cells, and F4/80(+) cells. However, MHC class II(+) passenger leukocytes in donor cornea after allografting did not traffic to the draining lymph node. Instead, Ag (plasmid) delivered to the eye via the donor cornea during allograft was detected in host CD11c(+) and F4/80(+) APC in the draining lymph nodes and spleen. The earliest detection of APC-associated Ag was at 6 h in the draining lymph node and 24 h in the spleen. After 48 h Ag was not detected in the draining lymph node but was still present in the spleen. Ag applied to the donor corneal epithelium before allografting induced Ag-specific T cell activation and expansion in the draining lymph node with a peak response at 4-6 days, indicating that cross-presentation of Ag had occurred. We conclude therefore, that Ag is transported from the donor cornea within host APC and that this event occurs within hours after grafting. Ag is cross-presented to host CD4(+) T cells on MHC class II and leads to the activation of Ag-specific effector T cells and clonal expansion in the draining lymph node.

Leukemic Blasts Acting as Host Antigen Presenting Cells Trigger a Combined CD4 and CD8 Allo-Immune Response Directed Against Mismatched HLA Class I.

Single HLA locus mismatched stem cell transplantation (SCT) is applied in patients with hematological malignancies who may benefit from allogeneic transplantation but lack an HLA-matched donor. Although HLA disparity between patient and donor increases the risk of developing GVHD, the relative risk of GVHD after single HLA locus mismatched SCT is only 1.5 fold. In view of the high frequency of allo-HLA reactive T-cells, which is about 1000-fold higher than the frequencies of minor histocompatibility antigen specific T-cells, this risk increase is lower than could be expected. Since almost all nucleated cells express HLA class I, one would expect all single HLA class I mismatched transplanted patients to develop severe GVHD. We hypothesized therefore that the presentation of the HLA class I mismatched allele on nucleated cells of the patient is not sufficient to elicit an effective allo-immune response. We characterized the allo-immune response in a patient with acute myeloid leukemia (AML) who was treated with a T-cell depleted SCT from a sibling donor who was HLA identical except for an HLA-A2 crossover. Six months after SCT, donor lymphocyte infusion (DLI) of 2.5*10e6 T-cells/kg was given for mixed chimerism comprising 99% T-cells of patient origin. No clinical response and no GVHD developed. Twelve months after SCT 95% of T-cells were still of patient origin, and AML relapse occurred with 9% blasts in bone marrow for which a second DLI containing 7.5*10e6 T-cells/kg was given. Five weeks after the DLI the patient died of grade IV GVHD. During the GVHD, conversion to donor chimerism developed. In peripheral blood of the patient 90% of CD8 and 40% of CD4 donor T-cells were activated as determined by HLA-DR expression. To analyze the nature of the immune response, the activated CD8 and CD4 donor T-cells were single cell sorted, expanded and tested for alloreactivity and HLA restriction using cytotoxicity and cytokine production assays against a panel of target cells blocked with different HLA-mAbs. 82% of the CD8 T-cell clones were alloreactive and restricted to the allo-HLA-A2. The response was highly polyclonal as shown by the usage of different T-cell receptor Vβ chains with different CDR3 sequences. 26% of the CD4 clones were alloreactive and this response was also polyclonal. The CD4 clones were HLA-DR1 restricted and recognized donor EBV-LCL transduced with HLA-A2, indicating that the peptide recognized in HLA-DR1 was derived from the mismatched HLA-A2 molecule. The recognized epitope was demonstrated to comprise AA 103–120 derived from a hypervariable region of HLA-A2. At the time of the first DLI, only HLA class I expressing T-cells and non-hematopoietic patient derived cells were present, capable of activating the CD8 T-cells but not of triggering the CD4 response. Leukemic blasts present at the time of the second DLI, however, expressed both HLA-DR and HLA class I, and were shown to activate the CD4 as well as the CD8 clones. We hypothesize that the HLA class II expression on hematopoietic cells of the patient at the time of the relapse was essential for the development of this immune response. In conclusion, these results indicate a role for patient leukemic blasts acting as host APCs in initiating the GVH response by activating both a CD4 and CD8 T-cell response in an HLA class I mismatched setting.

Effector and regulatory T-cell function is differentially regulated by RelB within antigen-presenting cells during GVHD

AbstractAntigen-presenting cells (APCs) are critical for the initiation of graft-versus-host disease (GVHD), although the responsible APC subset and molecular mechanisms remain unclear. Because dendritic cells (DCs) are the most potent APCs and the NF-kB/Rel family member RelB is associated with DC maturation and potent APC function, we examined their role in GVHD. Within 4 hours of total body irradiation, RelB nuclear translocation was increased and restricted to CD11chi DCs within the host APC compartment. Furthermore, the transient depletion of CD11chi donor DCs that reconstitute in the second week after transplantation resulted in a transient decrease in GVHD severity. By using RelB−/− bone marrow chimeras as transplant recipients or RelB−/− donor bone marrow, we demonstrate that the induction and maintenance of GVHD is critically dependent on this transcription factor within both host and donor APCs. Critically, RelB within APCs was required for the expansion of donor helper T cell type 1 (Th1) effectors and subsequent alloreactivity, but not the peripheral expansion or function of donor FoxP3+ regulatory T cells. These data suggest that the targeted inhibition of nuclear RelB translocation within APCs represents an attractive therapeutic strategy to dissociate effector and regulatory T-cell function in settings of Th1-mediated tissue injury.

Cutting Edge: Cross-Presented Intracranial Antigen Primes CD8+ T Cells

The CNS is considered immune privileged due to the blood-brain barrier and the absence of conventional lymphatics. Nonetheless, T cell immune responses specific for CNS Ag have been documented. Where these events are initiated and what cellular mechanisms are involved remain unknown. In this study, we established an experimental mouse model to evaluate the requirements for priming CD8+ T cells following the cross-presentation of intracranial Ag. Surprisingly, we find that even with a damaged blood-brain barrier, Ag presentation occurs in regional lymph nodes and not within the CNS itself. Only once the responding cells have expanded can they traffic to the site of CNS injury. Cross-presentation of intracranial Ag is efficient and the subsequent priming of CD8+ T cells is dependent on CD4+ T cell help and CD40 signaling in host APCs. Our findings have important implications for the initiation of T cell immune responses toward CNS Ags.

Peripheral Tolerance Induction Using Ethylenecarbodiimide-Fixed APCs Uses both Direct and Indirect Mechanisms of Antigen Presentation for Prevention of Experimental Autoimmune Encephalomyelitis

MHC class II (MHC II)-restricted T cell responses are a common driving force of autoimmune disease. Accordingly, numerous therapeutic strategies target CD4(+) T cells with the hope of attenuating autoimmune responses and restoring self-tolerance. We have previously reported that i.v. treatment with Ag-pulsed, ethylenecarbodiimide (ECDI)-fixed splenocytes (Ag-SPs) is an efficient protocol to induce Ag-specific tolerance for prevention and treatment of experimental autoimmune encephalomyelitis (EAE). Ag-SPs coupled with peptide can directly present peptide:MHC II complexes to target CD4(+) T cells in the absence of costimulation to induce anergy. However, Ag-SPs coupled with whole protein also efficiently attenuates Ag-specific T cell responses suggesting the potential contribution of alternative indirect mechanisms/interactions between the Ag-SPs and target CD4(+) T cells. Thus, we investigated whether MHC II compatibility was essential to the underlying mechanisms by which Ag-SP induces tolerance during autoimmune disease. Using MHC-deficient, allogeneic, and/or syngeneic donor Ag-SPs, we show that MHC compatibility between the Ag-SP donor and the host is not required for tolerance induction. Interestingly, we found that ECDI treatment induces apoptosis of the donor cell population which promotes uptake and reprocessing of donor cell peptides by host APCs resulting in the apparent MHC II-independent induction of tolerance. However, syngeneic donor cells are more efficient at inducing tolerance, suggesting that Ag-SPs induce functional Ag-SP tolerance via both direct and indirect (cross-tolerance) mechanisms leading to prevention and effective treatment of autoimmune disease.

320: In vivo activation of APCs with TLR ligands and tissue damage rather than amount of host APCs are critical factors that determine DLI-mediated GVL reactivity and GVHD in MHC-matched minor histocompatibility antigen (mHAg)-mismatched chimeras

We recently found that residual host CD11c+ DCs persist in the skin of MHC-matched chimeras after myeloablative conditioning and transplantation of T cell-repleted BM, and that those residual host APCs are insufficient for the induction of optimal DLI-mediated alloreactivity (J Immunol.,2006). Since the latter observation in established C3H.SW→B6 chimeras differs from the current paradigm in freshly irradiated MHC-matched chimeras, we hypothesized that in established chimeras, beside host APCs and donor T cells, the induction of DLI-mediated GVL reactivity and GVHD requires two additional factors: (a) TLR-mediated activation of APCs and (b) tissue damage.

Tumor endothelial marker 8 enhances tumor immunity in conjunction with immunization against differentiation Ag

We have previously shown that xenogeneic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8. In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma. TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anti-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models. A local immune response to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may alter T-cell interactions or homing. In this way, TEM8 may act more as an adjuvant than an immunologic target.

FTY720 enhances the activation‐induced apoptosis of donor T cells and modulates graft‐versus‐host disease

FTY720 is a novel immunosuppressant that improves the outcomes after solid organ and bone marrow transplantation (BMT) due to the sequestration of T cells into LN. We tested the hypothesis that the sequestration of donor T cells in LN by FTY720 would enhance their interaction with host APC, thus causing a greater degree of activation-induced apoptosis of alloreactive T cells, and thereby resulting in a reduction of graft-vs.-host disease (GVHD). The short-term administration of FTY720 improved the recipient survival after allogeneic BMT. FTY720 treatment facilitated a rapid contraction of the donor T cell pool in association with an increased degree of apoptosis of donor T cells. The donor T cell reactivity to host alloantigens was diminished in host's LN and adoptive transfer of donor T cells isolated from LN of FTY720-treated recipients of allogeneic BMT induced less severe GVHD in secondary recipients than the transfer from controls. Caspase-dependent apoptosis was involved in this mechanism because FTY720-induced protection was abrogated when a pan-caspase inhibitor was administered. These findings thus demonstrate the presence of a novel mechanism by which FTY720 modulates the allogeneic T cell responses: namely, by the induction of activation-induced apoptosis of alloreactive T cells in LN.

CD8+ but Not CD4+ T Cells Require Cognate Interactions with Target Tissues To Mediate GVHD across Only Minor H Antigens but CD4+ and CD8+ T Cells Both Require Direct Leukemic Contact for GVL.

There has been debate as to whether CD4+ or CD8+ T cells require direct cognate interactions with their tissue targets to mediate GVHD. In one report, GVHD mediated by CD4 or CD8 cells did not require target tissue MHCI or MHCII expression in MHC-disparate models (Teshima, et al 2002). However, GVHD lethality may have been due to cytokines produced by high-frequency alloreactive T cells primed by MHC-disparate host APCs. In contrast, MHC-matched, multiple minor H antigen (miHA) disparate GVHD mediated by CD4 or CD8 cells has been reported to be reduced or absent in host → donor chimeric recipients of a second transplant with donor BM and T cells. In this case, host antigen was restricted to hematopoietic cells and the authors concluded that tissue miHA expression must be required (Korngold and Sprent, 1982; Jones et al, 2003). However these experiments did not address whether T cells directly interacted with MHC on target tissues. Rather they demonstrated that a continuing source of host antigen was essential. To resolve this we created bone marrow chimeras in which hematopoiesis was wild type (wt) while the parenchyma was either MHC I- (B6→B6 beta-2-microglobulin−/− (β2M−/−)) or MHC II− (B6→B6 IAb−/−) and used these chimeras as recipients in GVHD-inducing second transplants. We found that B6→B6β2M−/− chimeras were completely resistant clinically and pathologically to CD8-mediated GVHD induced by a second transplant with C3H.SW (H-2b) BM and CD8+ T cells whereas control B6→B6 chimeras developed severe disease. Thus, CD8 cells require direct cognate interactions with target host tissues to induce MHC-matched, miHA disparate GVHD. In contrast, B6→B6 IAb−/− chimeras developed similar clinical and histologic GVHD (liver, ear, skin and bowel) as did control B6→B6 chimeras when retransplanted with 129/J (H-2b) bone marrow and purified CD4+ T cells. Notably we observed lymphocytic infiltrates in involved organs. Therefore, CD4 cells can mediate tissue damage without directly recognizing alloantigen presented by MHCII on target epithelial cells. This suggests an indirect mechanism, perhaps mediated by T cell release of factors after stimulation in tissues by donor-derived APCs presenting host antigens. Alternatively, donor CD4 cells may activate miHA-bearing macrophages to release inflammatory mediators. To investigate the requirement for cognate recognition in GVL, we created murine CML via retroviral-mediated bcr-abl (p210) transduction of bone marrow from wt B6, B6 IAb−/− and B6 β2M−/− mice. Using the C3H.SW→B6 and 129→B6 GVHD models we found that both CD8-and CD4-mediated GVL requires leukemic expression of MHCI and MHCII, respectively. Thus both CD8-mediated GVHD and GVL required cognate T cell:target interactions. However, for CD4 cells only GVL, but not GVHD required target cell MHCII expression. This indicates that CD4-mediated GVL and GVHD have distinct mechanisms of action. Further understanding of these may provide insight in how to deliver GVL with less GVHD.

CD4+CD25+ T Cells Can Inhibit CD8 T Cell Mediated GVHD: Requirement for In Vivo Recognition of Allogeneic Host MHC Class II Antigens.

CD4 regulatory T (Treg) cells have shown promise in the transplantation mileu including the ability to inhibit the development of graft vs host disease (GVHD) following allogeneic hematopoietic stem cell transplants (HCT). The antigen specificity of the Treg population(s) involved is not yet clear nor is the role of their activation following transplant. We are interested in determining the requirement for recognition of host MHC antigens following infusion of CD4+CD25+ T cells in an experimental model of GVHD. To clearly distinguish the requirements of regulatory vs GVH reactive cells, a model of CD8 T cell mediated GVHD was developed using highly purified BALB/c (H2d) donor CD8+ T cells (Miltenyi column, 95-98%). CD8 T cells were transplanted together with T cell depleted (TCD) BALB/c BMC into 12.0 GY (6.0 Gy split dose) TBI conditioned C57BL/6 (B6, H2b) recipients. To support development of GVHD by these cells, resistance was inhibited by treatment of recipients with anti-NK1.1mab (PK136) at Days -1, 0 and +7. BALB/c CD8+ T cells at doses of 5.0x106 but not 2.5x106 induced weight loss and some lethality in B6 recipients. 5x106 CD8+ T cells were then transplanted into B6-MHC class II−/ − recipients. GVHD symptoms including weight loss and lethality were readily apparent in these mice post-transplant. Interestingly, GVHD was consistently more severe with respect to the induction of weight loss and lethality in MHC Class II−/ − vs B6-wt recipients. Highly enriched BALB/c CD4+CD25+ T cells (> 95%) were produced from spleen and lymph node cells following negative (B-cells, CD8 and NK) and positive (CD25) selection using Miltenyi magnetic bead columns. Co-transplant of 1x106 CD4+CD25+ T cells together with BALB/c CD8+ T cells into B6 recipients inhibited GVHD as assessed by the absence of weight loss and lethality compared to B6 recipients of CD8+ T cells alone. In contrast, BALB/c CD4+CD25+ T cells failed to protect B6-MHC class II−/ − recipients from severe CD8+ T cell mediated GVHD. These findings demonstrate that donor CD4+ T regulatory cells can suppress GVHD inducing CD8+ T cells after the former recognize host class II alloantigen following transplant. We hypothesize that activated CD4+CD25+ T regulatory cells inhibit GVH reactive T cells at the host APC interface. Future studies in this model can be designed to examine ex-vivo activated and expanded CD4+CD25+ T regulatory populations. Transplant of such cells will enable us to address questions regarding the importance of in vivo recognition of host class II in the regulation of GVHD by these cells.

Respiratory Syncytial Virus Infection of Monocyte-Derived Dendritic Cells Decreases Their Capacity to Activate CD4 T Cells

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in children, the elderly, and immune-compromised individuals. CD4 and CD8 T cells play a crucial role in the elimination of RSV from the infected lung, but T cell memory is not sufficient to completely prevent reinfections. The nature of the adaptive immune response depends on innate immune reactions initiated after interaction of invading pathogens with host APCs. For respiratory pathogens myeloid dendritic cell (DC) precursors that are located underneath the epithelial cell layer lining the airways may play a crucial role in primary activation of T cells and regulating their functional potential. In this study, we investigated the role of human monocyte-derived DC in RSV infection. We showed that monocyte-derived DC can be productively infected, which results in maturation of the DC judged by the up-regulation of CD80, CD83, CD86, and HLA class II molecules. However, RSV infection of DC caused impaired CD4 T cell activation characterized by a lower T cell proliferation and ablation of cytokine production in activated T cells. The suppressive effect was caused by an as yet unidentified soluble factor produced by RSV-infected DC.

Both Dendritic Cells and Macrophages Can Stimulate Naive CD8 T Cells In Vivo to Proliferate, Develop Effector Function, and Differentiate into Memory Cells

The generation of T cell immunity requires the acquisition and presentation of Ag on bone marrow-derived APCs. Dendritic cells (DC) are believed to be the most potent bone marrow-derived APCs, and the only ones that can stimulate naive T cells to productively respond to Ags. Because macrophages (Mphi) are bone marrow-derived APCs that are also found in tissues and lymphoid organs, can acquire and present Ag, and can express costimulatory molecules, we have investigated their potential to stimulate primary T cell responses in vivo. We find that both injected Mphi and DCs can migrate from peripheral tissues or blood into lymphoid organs. Moreover, injection of peptide-pulsed Mphi or DCs into mice stimulates CD8 T cells to proliferate, express effector functions including cytokine production and cytolysis, and differentiate into long-lived memory cells. Mphi and DCs stimulate T cells directly without requiring cross-presentation of Ag on host APCs. Therefore, more than one type of bone marrow-derived APC has the potential to prime T cell immunity. In contrast, another bone marrow-derived cell, the T lymphocyte, although capable of presenting Ag and homing to the T cell areas of lymphoid organs, is unable to stimulate primary responses. Because Mphi can be very abundant cells, especially at sites of infection and inflammation, they have the potential to play an important role in immune surveillance and the initiation of T cell immunity.

Inefficient Cross-Presentation Limits the CD8+ T Cell Response to a Subdominant Tumor Antigen Epitope

CD8(+) T lymphocytes (T(CD8)) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote T(CD8) subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes T(CD8) specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce T(CD8) specific for the subdominant T Ag epitope V. Using adoptively transferred T(CD8) from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic T(CD8) in B6 mice required cross-presentation by host APC. However, robust expansion of these T(CD8) required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific T(CD8) contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting T(CD8) responses to cancer.