Efficient induction of CCR9 on T cells requires coactivation of retinoic acid receptors and retinoid X receptors (RXRs): exaggerated T Cell homing to the intestine by RXR activation with organotins

The active vitamin A metabolite retinoic acid (RA) imprints gut-homing specificity on lymphocytes upon activation by inducing the expression of α4β7 integrin and CCR9. RA receptor (RAR) activation is essential for their expression, whereas retinoid X receptor (RXR) activation is not essential for α4β7 expression. However, it remains unclear whether RXR activation affects the RA-dependent CCR9 expression on T cells and their gut homing. The major physiological RA, all-trans-RA, binds to RAR but not to RXR at physiological concentrations. Cell-surface CCR9 expression was often induced on a limited population of murine naive CD4(+) T cells by all-trans-RA or the RAR agonist Am80 alone upon CD3/CD28-mediated activation in vitro, but it was markedly enhanced by adding the RXR agonist PA024 or the RXR-binding environmental chemicals tributyltin and triphenyltin. Accordingly, CD4(+) T cells treated with the combination of all-trans-RA and tributyltin migrated into the small intestine upon adoptive transfer much more efficiently than did those treated with all-trans-RA alone. Furthermore, naive TCR transgenic CD4(+) T cells transferred into wild-type recipients migrated into the small intestinal lamina propria following i.p. injection of Ag, and the migration was enhanced by i.p. injection of PA024. We also show that PA024 markedly enhanced the all-trans-RA-induced CCR9 expression on naturally occurring naive-like regulatory T cells upon activation, resulting in the expression of high levels of α4β7, CCR9, and Foxp3. These results suggest that RXR activation enhances the RAR-dependent expression of CCR9 on T cells and their homing capacity to the small intestine.     

Flagellin, a TLR5 agonist, reduces graft-versus-host disease in allogeneic hematopoietic stem cell transplantation recipients while enhancing antiviral immunity

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). Posttransplant immunosuppressive drugs incompletely control GVHD and increase susceptibility to opportunistic infections. In this study, we used flagellin, a TLR5 agonist protein (～50 kDa) extracted from bacterial flagella, as a novel experimental treatment strategy to reduce both acute and chronic GVHD in allogeneic HSCT recipients. On the basis of the radioprotective effects of flagellin, we hypothesized that flagellin could ameliorate GVHD in lethally irradiated murine models of allogeneic HSCT. Two doses of highly purified flagellin (administered 3 h before irradiation and 24 h after HSCT) reduced GVHD and led to better survival in both H-2(b) → CB6F1 and H-2(K) → B6 allogeneic HSCT models while preserving >99% donor T cell chimerism. Flagellin treatment preserved long-term posttransplant immune reconstitution characterized by more donor thymic-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells and significantly enhanced antiviral immunity after murine CMV infection. The proliferation index and activation status of donor spleen-derived T cells and serum concentration of proinflammatory cytokines in flagellin-treated recipients were reduced significantly within 4 d posttransplant compared with those of the PBS-treated control recipients. Allogeneic transplantation of radiation chimeras previously engrafted with TLR5 knockout hematopoietic cells showed that interactions between flagellin and TLR5 expressed on both donor hematopoietic and host nonhematopoietic cells were required to reduce GVHD. Thus, the peritransplant administration of flagellin is a novel therapeutic approach to control GVHD while preserving posttransplant donor immunity.     

Post-BMT lung injury occurs independently of the expression of CCL2 or its receptor, CCR2, on host cells

Idiopathic pneumonia syndrome (IPS) is a significant cause of mortality post-bone marrow transplant (BMT) in humans. In our murine model, lethal pre-BMT conditioning and allogeneic T cells result in the recruitment of host antigen-presenting cells (APC) and donor T cells into the lung post-BMT concomitant with development of severe lung dysfunction. CCL2 induction is found in bronchoalveolar lavage fluid (BALF) before host monocyte influx. The major receptor for CCL2 is CCR2 present on monocytes; this interaction can play a crucial role in monocyte recruitment in inflammation. To determine whether blockade of the CCL2/CCR2 pathway could hinder host monocyte influx, lethally conditioned wild-type (WT), CCL2(-/-), or CCR2(-/-) mice were transplanted with allogeneic marrow and spleen cells. WT and (-/-) recipients exhibited equivalent lung dysfunction post-BMT. The frequencies of host macrophages as well as donor CD4(+) and CD8(+) T cells in lungs post-BMT did not differ between WT and (-/-) recipients. However, the T cell dependency of the host CD11b(+) major histocompatibility complex class II(+) cell influx was lost in CCR2(-/-) recipients. In CCR2(-/-) mice, this influx was accompanied by elevated levels of CCL20. Post-BMT BALF and sera of (-/-) mice did not reveal any decrease in cytokines or chemokines compared with WT mice. CCL2(-/-) mice had a deficiency of CCL2 in their BALF and sera post-BMT, confirming our hypothesis that CCL2 is predominantly host derived. Therefore, IPS can occur independently of host expression of CCL2 or CCR2, and compensatory mechanisms exist for regulating APC recruitment into the lung during the early post-BMT period.     

Trafficking of human immunodeficiency virus type 1-specific CD8+ T cells to gut-associated lymphoid tissue during chronic infection

Gut-associated lymphoid tissue (GALT) is a significant but understudied lymphoid organ, harboring a majority of the body's total lymphocyte population. GALT is also an important portal of entry for human immunodeficiency virus (HIV), a major site of viral replication and CD4(+) T-cell depletion, and a frequent site of AIDS-related opportunistic infections and neoplasms. However, little is known about HIV-specific cell-mediated immune responses in GALT. Using lymphocytes isolated from rectal biopsies, we have determined the frequency and phenotype of HIV-specific CD8(+) T cells in human GALT. GALT CD8(+) T cells were predominantly CD45RO(+) and expressed CXCR4 and CCR5. In 10 clinically stable, chronically infected individuals, the frequency of HIV Gag (SL9)-specific CD8(+) T cells was increased in GALT relative to peripheral blood mononuclear cells by up to 4.6-fold, while that of cytomegalovirus (CMV)-specific CD8(+) T cells was significantly reduced (P = 0.012). Both HIV- and CMV-specific CD8(+) T cells in GALT expressed CCR5, but only HIV-specific CD8(+) T cells expressed alpha E beta 7 integrin, suggesting that mucosal priming may account for their retention in GALT. Chronically infected individuals exhibited striking depletion of GALT CD4(+) T cells expressing CXCR4, CCR5, and alpha E beta 7 integrin, but CD4(+)/CD8(+) T-cell ratios in blood and GALT were similar. The percentage of GALT CD8(+) T cells expressing alpha E beta 7 was significantly decreased in infected individuals, suggesting that HIV infection may perturb lymphocyte retention in GALT. These studies demonstrate the feasibility of using tetramers to assess HIV-specific T cells in GALT and reveal that GALT is the site of an active CD8(+) T-cell response during chronic infection.     

Alloimmune response results in expansion of autoreactive donor CD4+ T cells in transplants that can mediate chronic graft-versus-host disease

Chronic graft-versus-host disease (cGVHD) is considered an autoimmune-like disease mediated by donor CD4(+) T cells, but the origin of the autoreactive T cells is still controversial. In this article, we report that the transplantation of DBA/2 donor spleen cells into thymectomized MHC-matched allogeneic BALB/c recipients induced autoimmune-like cGVHD, although not in control syngeneic DBA/2 recipients. The donor-type CD4(+) T cells from the former but not the latter recipients induced autoimmune-like manifestations in secondary allogeneic BALB/c as well as syngeneic DBA/2 recipients. Transfer of donor-type CD4(+) T cells from secondary DBA/2 recipients with disease into syngeneic donor-type or allogeneic host-type tertiary recipients propagated autoimmune-like manifestations in both. Furthermore, TCR spectratyping revealed that the clonal expansion of the autoreactive CD4(+) T cells in cGVHD recipients was initiated by an alloimmune response. Finally, hybridoma CD4(+) T clones derived from DBA/2 recipients with disease proliferated similarly in response to stimulation by syngeneic donor-type or allogeneic host-type dendritic cells. These results demonstrate that the autoimmune-like manifestations in cGVHD can be mediated by a population of donor CD4(+) T cells in transplants that simultaneously recognize Ags presented by both donor and host APCs.     

Naive and memory T cells induce different types of graft-versus-host disease

The goal of this study was to compare the ability of donor naive and alloantigen-primed effector memory T cells to induce graft-vs-host disease after bone marrow transplantation in MHC-mismatched irradiated host mice. Purified CD4(+) naive (CD62L(high)CD44(low)) T cells and CD4(+) effector memory (CD62L(low)CD44(high)) T cells obtained from unprimed donors and donors primed to host alloantigens, respectively, were injected into host mice, and the rapidity, severity, and pattern of tissue injury of graft-vs-host disease was assessed. Unexpectedly, the naive T cells induced a more acute and severe colitis than the primed memory cells. Whereas the naive T cells expressing CD62L and CCR7 lymph node homing receptors vigorously expanded in mesenteric lymph nodes and colon by day 6 after transplantation, the primed memory T cells without these receptors had 20- to 100-fold lower accumulation at this early time point. These differences were reflected in the significantly more rapid decline in survival and weight loss induced by naive T cells. The primed memory T cells had a greater capacity to induce chronic colitis and liver injury and secrete IL-2 and IFN-gamma in response to alloantigenic stimulation compared with memory T cells from unprimed donors. Nevertheless, the expected increase in potency as compared with naive T cells was not observed due to differences in the pattern and kinetics of tissue injury.     

Differing requirements for CCR4, E-selectin, and α4β1 for the migration of memory CD4 and activated T cells to dermal inflammation

CCR4 on T cells is suggested to mediate skin homing in mice. Our objective was to determine the interaction of CCR4, E-selectin ligand (ESL), and α(4)β(1) on memory and activated T cells in recruitment to dermal inflammation. mAbs to rat CCR4 were developed. CCR4 was on 5-21% of memory CD4 cells, and 20% were also ESL(+). Anti-TCR-activated CD4 and CD8 cells were 40-55% CCR4(+), and ～75% of both CCR4(+) and CCR4(-) cells were ESL(+). CCR4(+) memory CD4 cells migrated 4- to 7-fold more to dermal inflammation induced by IFN-γ, TNF, TLR agonists, and delayed-type hypersensitivity than CCR4(-) cells. CCR4(+) activated CD4 cells migrated only 5-50% more than CCR4(-) cells to these sites. E-selectin blockade inhibited ～60% of CCR4(+) activated CD4 cell migration but was less effective on memory cells where α(4)β(1) was more important. Anti-α(4)β(1) also inhibited CCR4(-) activated CD4 cells more than CCR4(+) cells. Anti-E-selectin reduced activated CD8 more than CD4 cell migration. These findings modify our understanding of CCR4, ESL, α(4)β(1), and dermal tropism. There is no strict relationship between CCR4 and ESL for skin homing of CD4 cells, because the activation state and inflammatory stimulus are critical determinants. Dermal homing memory CD4 cells express CCR4 and depend more on α(4)β(1) than ESL. Activated CD4 cells do not require CCR4, but CCR4(+) cells are more dependent on ESL than on α(4)β(1), and CCR4(-) cells preferentially use α(4)β(1). The differentiation from activated to memory CD4 cells increases the dependence on CCR4 for skin homing and decreases the requirement for ESL.     

Dysfunction of Bone Marrow Vascular Niche in Acute Graft-Versus-Host Disease after MHC-Haploidentical Bone Marrow Transplantation

Acute graft-versus-host disease (aGvHD) is the most common complication of allogeneic hematopoietic stem cell transplantation (HSCT), which is often accompanied by impaired hematopoietic reconstitution. Sinusoidal endothelial cells (SECs) constitute bone marrow (BM) vascular niche that plays an important role in supporting self-renewal capacity and maintaining the stability of HSC pool. Here we provide evidences that vascular niche is a target of aGvHD in a major histocompatibility complex (MHC)–haploidentical matched murine HSCT model. The results demonstrated that hematopoietic cells derived from GvHD mice had the capacity to reconstitute hematopoiesis in healthy recipient mice. However, hematopoietic cells from healthy donor mice failed to reconstitute hematopoiesis in GvHD recipient mice, indicating that the BM niche was impaired by aGvHD in this model. We further demonstrated that SECs were markedly reduced in the BM of aGvHD mice. High level of Fas and caspase-3 expression and high rate of apoptosis were identified in SECs, indicating that SECs were destroyed by aGvHD in this murine HSCT model. Furthermore, high Fas ligand expression on engrafted donor CD4⁺, but not CD8⁺ T cells, and high level MHC-II but not MHC-I expression on SECs, suggested that SECs apoptosis was mediated by CD4⁺ donor T cells through the Fas/FasL pathway.     

Mechanisms of Cyclic Nucleotide Phosphodiesterases in Modulating T Cell Responses in Murine Graft-versus-Host Disease

Graft-versus-host disease (GvHD) is a key contributor to the morbidity and mortality after allogeneic hematopoetic stem cell transplantation (HSCT). Regulatory Foxp3⁺ CD4⁺ T cells (T_reg) suppress conventional T cell activation and can control GvHD. In our previous work, we demonstrate that a basic mechanism of T_reg mediated suppression occurs by the transfer of cyclic adenosine monophosphate (cAMP) to responder cells. Whether this mechanism is relevant for T_reg mediated suppression of GvHD is currently unknown. To address this question, bone marrow and T cells from C57BL/6 mice were transferred into lethally irradiated BALB/c recipients, and the course of GvHD and survival were monitored. Transplanted recipients developed severe GvHD that was strongly ameliorated by the transfer of donor T_reg cells. Towards the underlying mechanisms, in vitro studies revealed that T_reg communicated with DCs via gap junctions, resulting in functional inactivation of DC by a metabolic pathway involving cAMP that is modulated by the phosphodiesterase (PDE) 4 inhibitor rolipram. PDE2 or PDE3 inhibitors as well as rolipram suppressed allogeneic T cell activation, indirectly by enhancing T_reg mediated suppression of DC activation and directly by inhibiting responder T cell proliferation. In line with this, we observed a cooperative suppression of GvHD upon T_reg transfer and additional rolipram treatment. In conclusion, we propose that an important pathway of T_reg mediated control of GvHD is based on a cAMP dependent mechanism. These data provide the basis for future concepts to manipulate allogeneic T cell responses to prevent GvHD.     

Multilineage engraftment with minimal graft-versus-host disease following in utero transplantation of S-59 psoralen/ultraviolet a light-treated,sensitized T cells and adult T cell-depleted bone marrow in fetal mice

Although engraftment following in utero stem cell transplantation can readily be achieved, a major limitation is the low level of donor chimerism. We hypothesized that a lack of space for donor cells in the recipient marrow was one of the primary reasons for failure to achieve significant engraftment, and that donor T cells could make space in an allogeneic mismatched setting. We found that 3 x 10(5) C57BL/6 (B6) naive CD3(+) cells coinjected with B6 T cell-depleted bone marrow (TCDBM) into 14- to 15-day-old BALB/c fetuses resulted in multilineage engraftment (median, 68.3%) associated with severe graft-vs-host disease (GvHD; 62 vs 0% with TCDBM alone). When 1.5 x 10(5) CD4(+) or CD8(+) cells were used, low levels of engraftment were seen vs recipients of 1.5 x 10(5) CD3(+) cells (2.4 +/- 1.1 and 6.6 +/- 3.9 vs 20.4 +/- 10.4%, respectively). To test the hypothesis that proliferation of T cells in response to alloantigen resulted in GvHD and increased engraftment, we pretreated naive T cells with photochemical therapy (PCT) using S-59 psoralen and UVA light to prevent proliferation. GvHD was reduced (60-0%), but was also associated with a significant reduction in engrafted donor cells (53.4 +/- 4.2 to 1.7 +/- 0.5%). However, when B6 T cells were sensitized to BALB/c splenocytes, treated with PCT, and coinjected with TCDBM, there was a partial restoration of engraftment (13.3 +/- 2.4% H2Kb(+) cells) with only one of nine animals developing mild to moderate GvHD. In this study we have shown that PCT-treated T cells that are cytotoxic but nonproliferative can provide an engraftment advantage to donor cells, presumably by destroying host hemopoietic cells without causing GvHD.     

G-CSF-treated donor CD4(+) T cells attenuate acute GVHD through a reduction in Th17 cell differentiation

The immunoregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic peripheral blood cell transplantation (PBCT) have been demonstrated to reduce acute graft-versus-host disease (GVHD). However, the underlying mechanism is still not clear. In this study, we focused on the direct effects of G-CSF on donor CD4(+) T cell responses after transplantation. We observed that lethally irradiated B6D2F1 recipient mice that are transplanted with CD4(+) T cells from G-CSF-treated B6 donors showed mild attenuations in severity and mortality compared with recipients transplanted with PBS-treated CD4(+) T cells. Notably, skin GVHD was significantly reduced, but no such reduction was observed in other organs. Although there was no difference with respect to alloreactive expansion or Foxp3(+) Treg induction, the use of G-CSF-treated CD4(+) T cells significantly reduced the numbers of IL-17-producing and RORγt-expressing cells in the secondary lymphoid organs of allogeneic recipients after transplantation compared with the use of the control cells. Finally, we found that the suppressor of cytokine signaling-3 (SOCS3) expression in G-CSF-treated donor CD4(+) T cells was much higher than that in control CD4(+) T cells. Our results demonstrate that the inhibition of Th17 cell differentiation by SOCS3 induction is associated with the immunoregulatory role of G-CSF in CD4(+) T cell-mediated acute GVHD.     

Monitoring of human cytomegalovirus and virus-specific T-cell response in young patients receiving allogeneic hematopoietic stem cell transplantation

In allogeneic hematopoietic stem-cell transplantation (HSCT) recipients, outcome of human cytomegalovirus (HCMV) infection results from balance between viral load/replication and pathogen-specific T-cell response. Using a cut-off of 30,000 HCMV DNA copies/ml blood for pre-emptive therapy and cut-offs of 1 and 3 virus-specific CD4(+) and CD8(+) T cells/μl blood for T-cell protection, we conducted in 131 young patients a prospective 3-year study aimed at verifying whether achievement of such immunological cut-offs protects from HCMV disease. In the first three months after transplantation, 55/89 (62%) HCMV-seropositive patients had infection and 36/55 (65%) were treated pre-emptively, whereas only 7/42 (17%) HCMV-seronegative patients developed infection and 3/7 (43%) were treated. After 12 months, 76 HCMV-seropositive and 9 HCMV-seronegative patients (cumulative incidence: 90% and 21%, respectively) displayed protective HCMV-specific immunity. Eighty of these 85 (95%) patients showed spontaneous control of HCMV infection without additional treatment. Five patients after reaching protective T-cell levels needed pre-emptive therapy, because they developed graft-versus-host disease (GvHD). HSCT recipients reconstituting protective levels of HCMV-specific T-cells in the absence of GvHD are no longer at risk for HCMV disease, at least within 3 years after transplantation. The decision to treat HCMV infection in young HSCT recipients may be taken by combining virological and immunological findings.     

Differential roles for CCR5 expression on donor T cells during graft-versus-host disease based on pretransplant conditioning

The coordinated expression of chemokines and receptors may be important in the directed migration of alloreactive T cells during graft-vs-host disease (GVHD). Recent work demonstrated in a murine model that transfer of CCR5-deficient (CCR5(-/-)) donor cells to nonconditioned haploidentical recipients resulted in reduced donor cell infiltration in liver and lymphoid tissues compared with transfer of CCR5(+/+) cells. To investigate the function of CCR5 during GVHD in conditioned transplant recipients, we transferred CCR5(-/-) or wild-type C57BL/6 (B6) T cells to lethally irradiated B6D2 recipients. Unexpectedly, we found an earlier time to onset and a worsening of GVHD using CCR5(-/-) T cells, which was associated with significant increases in the accumulation of alloreactive CD4(+) and CD8(+) T cells in liver and lung. Conversely, the transfer of CCR5(-/-) donor cells to nonirradiated recipients led to reduced infiltration of target organs, confirming previous studies and demonstrating that the role of CCR5 on donor T cells is dependent on conditioning of recipients. Expression of proinflammatory chemokines in target tissues was dependent on conditioning of recipients, such that CXCL10 and CXCL11 were most highly expressed in tissues of irradiated recipients during the first week post-transplant. CCR5(-/-) T cells were shown to have enhanced migration to CXCL10, and blocking this ligand in vivo improved survival in irradiated recipients receiving CCR5(-/-) T cells. Our data indicate that the effects of inhibiting CCR5/ligand interaction on donor T cells during GVHD differ depending on conditioning of recipients, a finding with potentially important clinical significance.     

APCs in the liver and spleen recruit activated allogeneic CD8+ T cells to elicit hepatic graft-versus-host disease

Host APCs are required for initiating T cell-dependent acute graft-vs-host disease (GVHD), but the role of APCs in the effector phase of acute GVHD is not known. To measure the effect of tissue-resident APCs on the local development of acute GVHD, we selectively depleted host macrophages and DCs from the livers and spleens, but not from the skin, peripheral lymph nodes (PLN), or mesenteric lymph nodes (MLN), of C57BL/6 (B6) mice by i.v. administration of liposomal clodronate before allogeneic bone marrow transplantation. Depletion of host hepatic and splenic macrophages and DCs significantly inhibited the proliferation of donor C3H.SW CD8(+) T cells in the spleen, but not in the PLN or MLN, of B6 mice. Such organ-selective depletion of host tissue APCs also markedly reduced the trafficking of allogeneic CD8(+) T cells into the livers and spleens, but not PLN and MLN, of B6 recipients compared with that of the control mice. Acute hepatic, but not cutaneous, GVHD was inhibited as well, resulting in improved survival of liposomal clodronate-treated B6 recipients. When C3H.SW CD8(+) T cells were activated in normal B6 recipients, recovered, and adoptively transferred into secondary B6 recipients, activated donor CD8(+) T cells rapidly migrated into the livers and spleens of control B6 recipients but were markedly decreased in B6 mice that were depleted of hepatic and splenic macrophages and DCs. Thus, tissue-resident APCs control the local recruitment of allo-reactive donor T cells and the subsequent development of acute GVHD.     

Alloreactive memory T cells are responsible for the persistence of graft-versus-host disease

Graft-vs-host disease (GVHD) is caused by a donor T cell anti-host reaction that evolves over several weeks to months, suggesting a requirement for persistent alloreactive T cells. Using the C3H.SW anti-C57BL/6 (B6) mouse model of human GVHD directed against minor histocompatibility Ags, we found that donor CD8(+) T cells secreting high levels of IFN-gamma in GVHD B6 mice receiving C3H.SW naive CD8(+) T cells peaked by day 14, declined by day 28 after transplantation, and persisted thereafter, corresponding to the kinetics of a memory T cell response. Donor CD8(+) T cells recovered on day 42 after allogeneic bone marrow transplantation expressed the phenotype of CD44(high)CD122(high)CD25(low), were able to homeostatically survive in response to IL-2, IL-7, and IL-15 and rapidly proliferated upon restimulation with host dendritic cells. Both allogeneic effector memory (CD44(high)CD62L(low)) and central memory (CD44(high)CD62L(high)) CD8(+) T cells were identified in B6 mice with ongoing GVHD, with effector memory CD8(+) T cells as the dominant (>80%) population. Administration of these allogeneic memory CD8(+) T cells into secondary B6 recipients caused virulent GVHD. A similar allogeneic memory CD4(+) T cell population with the ability to mediate persistent GVHD was also identified in BALB/b mice receiving minor histocompatibility Ag-mismatched B6 T cell-replete bone marrow transplantation. These results indicate that allogeneic memory T cells are generated in vivo during GVH reactions and are able to cause GVHD, resulting in persistent host tissue injury. Thus, in vivo blockade of both alloreactive effector and memory T cell-mediated host tissue injury may prove to be valuable for GVHD prevention and treatment.     

Functional expression of chemokine receptor CCR5 on CD4(+) T cells during virus-induced central nervous system disease

Intracranial infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a demyelinating disease similar in pathology to the human disease multiple sclerosis (MS). CD4(+) T cells are important in amplifying demyelination by attracting macrophages into the central nervous system (CNS) following viral infection; however, the mechanisms governing the entry of these cells into the CNS are poorly understood. The role of chemokine receptor CCR5 in trafficking of virus-specific CD4(+) T cells into the CNS of MHV-infected mice was investigated. CD4(+) T cells from immunized CCR5(+/+) and CCR5(-/-) mice were expanded in the presence of the immunodominant epitope present in the MHV transmembrane (M) protein encompassing amino acids 133 to 147 (M133-147). Adoptive transfer of CCR5(+/+)-derived CD4(+) T cells to MHV-infected RAG1(-/-) mice resulted in CD4(+)-T-cell entry into the CNS and clearance of virus from the brain. These mice also displayed robust demyelination correlating with macrophage accumulation within the CNS. Conversely, CD4(+) T cells from CCR5(-/-) mice displayed an impaired ability to traffic into the CNS of MHV-infected RAG1(-/-) recipients, which correlated with increased viral titers, diminished macrophage accumulation, and limited demyelination. Analysis of chemokine receptor mRNA expression by M133-147-expanded CCR5(-/-)-derived CD4(+) T cells revealed reduced expression of CCR1, CCR2, and CXCR3, indicating that CCR5 signaling is important in increased expression of these receptors, which aid in trafficking of CD4(+) T cells into the CNS. Collectively these results demonstrate that CCR5 signaling is important to migration of CD4(+) T cells to the CNS following MHV infection.     

DNA immunization against tissue-restricted antigens enhances tumor immunity after allogeneic hemopoietic stem cell transplantation

Malignant relapse remains a major problem for recipients of allogeneic hemopoietic stem cell transplantation (HSCT). We hypothesized that immunization of allogeneic HSCT recipients against tissue-restricted Ags using DNA vaccines would decrease the risk of relapse without enhancing graft-vs-host disease (GVHD). Using the mouse B16 melanoma model, we found that post-HSCT DNA immunization against a single tumor Ag induces tumor rejection that is significantly greater than HSCT alone in a T cell-depleted MHC-matched minor Ag-mismatched allogeneic HSCT model (LP --> B6). In treatment models, post-HSCT DNA immunization provides significantly greater overall survival than the vaccine alone. Donor leukocyte infusion further enhances tumor-free survival, including in treatment models. There was no GVHD in HSCT recipients treated with DNA vaccination and donor leukocyte infusion. Further analysis demonstrated that these effects are dependent on CD8+ T cells of donor origin that recognize multiple epitopes. These results demonstrate that DNA immunization against tissue-restricted Ags after allogeneic T cell-depleted HSCT can induce potent antitumor effects without causing GVHD.     

Human bone marrow hosts polyfunctional memory CD4+ and CD8+ T cells with close contact to IL-15-producing cells

Recently, a key role in memory T cell homing and survival has been attributed to the bone marrow (BM) in mice. In the human BM, the repertoire, function, and survival niches of CD4(+) and CD8(+) T cells have not yet been elucidated. In this study, we demonstrate that CD4(+) and CD8(+) effector memory T cells accumulate in the human BM and are in a heightened activation state as revealed by CD69 expression. BM-resident memory T cells produce more IFN-γ and are frequently polyfunctional. Immunofluorescence analysis revealed that CD4(+) and CD8(+) T cells are in the immediate vicinity of IL-15-producing BM cells, suggesting a close interaction between these two cell types and a regulatory role of IL-15 on T cells. Accordingly, IL-15 induced an identical pattern of CD69 expression in peripheral blood CD4(+) and CD8(+) T cell subsets. Moreover, the IL-15-inducible molecules Bcl-x(L), MIP-1α, MIP-1β, and CCR5 were upregulated in the human BM. In summary, our results indicate that the human BM microenvironment, in particular IL-15-producing cells, is important for the maintenance of a polyfunctional memory CD4(+) and CD8(+) T cell pool.     

Allogeneic bone marrow grafts with high levels of CD4(+) CD25(+) FoxP3(+) T cells can lead to engraftment failure

Regulatory CD4(+) CD25(+) FoxP3(+) T cells (T(regs) ) suppress immunological reactions. However, the effect of adding T(regs) to hematopoietic stem cell grafts on recovery and graft versus host disease (GvHD) is unknown. T(regs) from splenocytes of C57Bl/6 and Balb/c wild-type mice were isolated by MACS separation and analyzed by flow cytometry. Using a murine syngeneic transplantation model that clearly distinguishes between donor and host hematopoiesis, we showed that co-transplantation of bone marrow cells (BMCs) with high levels of T(regs) leads to a 100% survival of the mice and accelerates the hematopoietic recovery significantly (full donor chimerism). In allogeneic transplantation, bone marrow and T(regs) co-transplantation were compared to allogeneic bone marrow transplantation with or without the addition of splenocytes. Survival, leukocyte recovery, chimerism at days -2, 19, 33, and 61 for murine CD4, human CD4, HLA-DR3, murine CD3, murine CD8, murine Balb/c-H2K(d) , murine C57Bl/6-H2K(b) , and GvHD appearance were analyzed. Allogeneic bone marrow transplantation requires the addition of splenocytes to reach engraftment. Mice receiving grafts with bone marrow, splenocytes and high levels of allogeneic T(regs) died within 28 days (hematopoietic failure). Here, we show also detailed flow cytometric data reagarding analysis of chimerism after transplantation in unique murine hematopoietic stem cell transplantation models. Our findings showed that the syngeneic co-transplantation of CD4(+) , CD25(+) , FoxP3(+) T-cells and BMCs induced a stimulating effect on reconstitution of hematopoiesis after irradiation. However, in the allogeneic setting the co-transplantation of T(regs) aggravates the engraftment of transplanted cells.