The therapeutic success of adoptive therapy with chimeric antigen receptor (CAR) engineered T cells depends on the appropriate costimulation of CD3ζ to induce full T cell activation. Costimulatory endodomains of the CD28 family are therefore fused with CD3ζ in a dual signalling CAR. Serious adverse events in two most recent trials; however, highlight the need to analyse in more detail the impact of each costimulatory endodomain on individual effector functions of redirected T cells. We therefore performed a thoroughly controlled side-by-side comparison of the most frequently used endodomains with respect to their impact on CD4(+) and CD8(+) T cell effector functions. CD28 reinforced T cell proliferation and is mandatory to induce IL-2. In the absence of added IL-2, CD28 and OX40 (CD137) but not 4-1BB (CD134) enhanced specific cytolysis. While CD28, 4-1BB and OX40 similarly improved pro-inflammatory cytokine secretion, OX40 most efficiently prevented activation induced cell death of CD62L(-) effector memory T cells. CD28 was superior to initiate the T cell response, OX40 and 4-1BB sustained the response in long term with OX40 being most effective. We consequently combined the beneficial functions in a 3rd generation CD28-OX40 CAR which substantially improved the antitumor response without loosing specificity.

Cutaneous (CL) and mucosal leishmaniasis (ML) are characterized by a predominant type 1 immune response (IFN-gamma and TNF-alpha production) and strong inflammatory response in the lesions with few parasites. This exacerbated type 1 response is more evident in ML as compared to CL. Our main hypothesis is that a differential immune regulation of T cell activation leads to over reactive T cells in ML. In the present study, we investigated immunological factors that could explain the mechanisms behind it by comparing some immune regulatory mechanisms between ML and CL patients: frequency of cells expressing co-stimulatory molecules, apoptotic markers, T cell activation markers; and ability of neutralizing antibodies to IL-2, IL-12 and IL-15 do down-regulate IFN-gamma production in leishmania antigen-stimulated peripheral blood mononuclear cells (PBMC). Interestingly, in CL anti-IL-2 and anti-IL-15 significantly suppressed antigen-specific IFN-gamma production, while in ML only anti-IL-2 suppressed IFN-gamma production. Finally, higher frequency of CD4+ T cells expressing CD28-, CD69+ and CD62L(low) were observed in ML as compared to CL. These data indicate that an exacerbated type 1 response in ML is differentially regulated and not appropriately down modulated, with increased frequencies of activated effectors T cells, maintaining the persistent inflammatory response and tissue damage observed in ML.

BACKGROUND

Regulatory T cells (Treg) play important roles in preventing autoimmunity, graft-versus host disease and transplant rejection. In rodent transplant models, tolerance induction strategies can induce graft protective CD25CD4 Treg in vivo but therapeutic exploitation of active regulation will more likely depend on protocols that allow generation or selection of regulatory cells ex vivo for use as a cellular therapy. We have used adoptive transfer skin and islet allograft models to identify, develop and evaluate ex vivo protocols that generate donor-reactive, adaptive Treg.

METHODS

Naïve CDA CD4 T cells were stimulated with allogeneic antigen-presenting cell under neutral conditions or with cytokine modification, restimulated under identical conditions and subsequently analyzed for cytokine profile, phenotypic markers characteristic of Treg and in vivo regulatory function.

RESULTS

Without modification, CD4 T cells default to a Th2 phenotype characterized by a dominant interleukin-4 response which is profoundly detrimental to allograft survival. However, addition of exogenous interferon-gamma suppresses interleukin-4 production without priming for effector function, induces suppressor of cytokine signaling-1 and results in up-regulation of Foxp3 and CD62L. The generation of these populations is enhanced by, but is independent of, the presence of naturally occurring endogenous Treg. Most importantly, when tested for regulatory function in vivo, these cells prevent rejection of both skin and islet allografts mediated by effector T cells.

CONCLUSIONS

These data reveal an unexpected role for interferon-gamma in the generation of Treg ex vivo and suggest a possible route for the generation of regulatory cells for therapeutic use.

OBJECTIVE

Experimental and clinical studies support a role for the fetus in the control of the onset of labor. Fetal systemic inflammation, but not a maternal inflammatory response, has been linked to the onset of preterm labor and delivery on the basis of the determination of inflammatory cytokines in fetal and maternal blood. We propose that parturition requires fetomaternal cooperation and that inflammation is an integral part of the parturitional process. This study used flow cytometry, a sensitive technique for the detection of intravascular inflammation, to assess whether maternal inflammation is present in preterm labor.

METHODS

A prospective cross-sectional study was performed including patients with preterm labor (n = 55) and women with normal pregnancy (n = 50). Intravascular inflammation was studied by using flow cytometry. Maternal blood was assayed to determine granulocyte and monocyte phenotype by using monoclonal antibodies, which included the following cluster of differentiation (CD) markers: CD11b, CD14, CD15, CD16, CD18, CD49d, CD62L, CD64, CD66b, and HLA-DR. Oxidative burst and generation of basal intracellular oxygen radical species were assessed. Statistical analysis was conducted with the use of nonparametric methods. A P value of <.01 was considered statistically significant.

RESULTS

Preterm labor was associated with a significant increase in the median mean channel brightness of CD11b, CD15, and CD66b on granulocytes and median mean channel brightness of CD11b and CD15 on monocytes. The ratio of oxidative burst over basal intracellular oxygen radical species in both granulocytes and monocytes was increased in preterm labor (P <. 01).

CONCLUSIONS

Preterm labor with intact membranes is associated with phenotypic and metabolic changes of maternal granulocytes and monocytes.

Peripheral blood and intestinal CD4+CD8+ double-positive (DP) T cells have been described in several species including humans, but their function and immunophenotypic characteristics are still not clearly understood. Here we demonstrate that DP T cells are abundant in the intestinal lamina propria of normal rhesus macaques (Macaca mulatta). Moreover, DP T cells have a memory phenotype and are capable of producing different and/or higher levels of cytokines and chemokines in response to mitogen stimulation compared to CD4+ single-positive T cells. Intestinal DP T cells are also highly activated and have higher expression of CCR5, which makes them preferred targets for simian immunodeficiency virus/HIV infection. Increased levels of CD69, CD25 and HLA-DR, and lower CD62L expression were found on intestinal DP T cells populations compared to CD4+ single-positive T cells. Collectively, these findings demonstrate that intestinal and peripheral blood DP T cells are effector cells and may be important in regulating immune responses, which distinguishes them from the immature DP cells found in the thymus. Finally, these intestinal DP T cells may be important target cells for HIV infection and replication due to their activation, memory phenotype and high expression of CCR5.

Expression of the cell-surface antigen CD10 has long been used to define the lymphoid commitment of human cells. Here we report a unique lymphoid-primed population in human bone marrow that was generated from hematopoietic stem cells (HSCs) before onset of the expression of CD10 and commitment to the B cell lineage. We identified this subset by high expression of the homing molecule L-selectin (CD62L). CD10(-)CD62L(hi) progenitors had full lymphoid and monocytic potential but lacked erythroid potential. Gene-expression profiling placed the CD10(-)CD62L(hi) population at an intermediate stage of differentiation between HSCs and lineage-negative (Lin(-)) CD34(+)CD10(+) progenitors. CD62L was expressed on immature thymocytes, and its ligands were expressed at the cortico-medullary junction of the thymus, which suggested a possible role for this molecule in homing to the thymus. Our studies identify the earliest stage of lymphoid priming in human bone marrow.

Bcl6 plays a role in the generation and maintenance of memory CD8(+) T cells. We analyzed here a role for Bcl6 in the generation of long-term memory CD4(+) T cells. Naive CD45RB(+) CD4(+) T cells from Bcl6-deficient DO11.10 (KJ1.26(+)) transgenic mice were transferred into BALB/c mice and immunized with ovalbumin peptide and LPS. Long-term memory KJ1.26(+) CD4(+) T cells from wild-type mice were detected in the spleen, lungs and liver during 10 weeks after immunization; however, Bcl6-deficient KJ1.26(+) CD4(+) T cells were vanished completely in those organs 4 weeks after immunization. Since memory CD4(+) T cells can be generated from effector CD4(+) T cells, properties of Bcl6-deficient effector CD4(+) T cells were compared with those wild-type effector CD4(+) T cells 10 days after immunization. Numbers of IFN-gamma-non-producing CD45RB(-), CD62L(+) or IL-7Ralpha(+) effector CD4(+) T cells in the spleen, lungs and liver were similar between Bcl6-deficient and wild-type CD4(+) T cells. However, the percentage of apoptotic cells in Bcl6-deficient effector CD4(+) T cells was higher than that in wild-type effector CD4(+) T cells. At the late effector phase, the number of IFN-gamma-non-producing cells and the percentage of apoptotic cells in Bcl6-deficient CD4(+) T cells were smaller and higher than those in wild-type CD4(+) T cells, respectively. These data suggest that Bcl6 in CD4(+) T cells plays a role in protection of memory precursor CD4(+) T cells from apoptosis and may involve in survivability of long-term memory CD4(+) T cells.

Diabetes compromises the bone marrow (BM) microenvironment and reduces the number of circulating CD34(+) cells. Diabetic autonomic neuropathy (DAN) may impact the BM, because the sympathetic nervous system is prominently involved in BM stem cell trafficking. We hypothesize that neuropathy of the BM affects stem cell mobilization and vascular recovery after ischemia in patients with diabetes. We report that, in patients, cardiovascular DAN was associated with fewer circulating CD34(+) cells. Experimental diabetes (streptozotocin-induced and ob/ob mice) or chemical sympathectomy in mice resulted in BM autonomic neuropathy, impaired Lin(-)cKit(+)Sca1(+) (LKS) cell and endothelial progenitor cell (EPC; CD34(+)Flk1(+)) mobilization, and vascular recovery after ischemia. DAN increased the expression of the 66-kDa protein from the src homology and collagen homology domain (p66Shc) and reduced the expression of sirtuin 1 (Sirt1) in mice and humans. p66Shc knockout (KO) in diabetic mice prevented DAN in the BM, and rescued defective LKS cell and EPC mobilization. Hematopoietic Sirt1 KO mimicked the diabetic mobilization defect, whereas hematopoietic Sirt1 overexpression in diabetes rescued defective mobilization and vascular repair. Through p66Shc and Sirt1, diabetes and sympathectomy elevated the expression of various adhesion molecules, including CD62L. CD62L KO partially rescued the defective stem/progenitor cell mobilization. In conclusion, autonomic neuropathy in the BM impairs stem cell mobilization in diabetes with dysregulation of the life-span regulators p66Shc and Sirt1.

BACKGROUND

Multiple myeloma (MM) is an immunoproliferative disease characterised by the uncontrolled proliferation of plasma cells, which is accompanied by defects in the immune system.

METHODS

This study aimed to characterise the frequency of T regulatory cells (Tregs), dendritic cells (DCs) as well as sub-populations of T cells bearing regulatory properties like CD4(+)GITR(+), CD4(+)CD62L(+), CD3(+)TCRγδ(+) along with the concentrations of IL-10, TGFβ, IL-6 in 66 patients with MM. Subsequently, the influence of therapy on those components of immune system was assessed.

RESULTS

The percentage of both myeloid and plasmacytoid DC was lower in MM compared with control group while Treg (CD4(+)CD25(high)FOXP3(+)) frequencies were significantly higher in MM patients compared with healthy control (6.16% vs 0.05%, respectively). Also, the percentages of CD4(+)GITR(+), CD4(+)CD62L(+) were increased compared with healthy volunteers. We found that patients with higher percentages of Treg live shorter (median overall survival 21 months vs not-reached, P=0.013).

CONCLUSIONS

This study identifies several abnormalities of immune system in MM, which only partly could be normalised after successful therapy. The dysfunction of immune system such as decreased antigen presentation along with increased frequencies of suppressive cells and cytokines might facilitate progression of the disease and infectious complications limiting survival of MM patients.

Clearance of disseminated Salmonella infection requires bacterial-specific Th1 cells and IFN-γ production, and Th1-promoting vaccines are likely to help control these infections. Consequently, vaccine design has focused on developing Th1-polarizing adjuvants or Ag that naturally induce Th1 responses. In this study, we show that, in mice, immunization with soluble, recombinant FliC protein flagellin (sFliC) induces Th2 responses as evidenced by Ag-specific GATA-3, IL-4 mRNA, and protein induction in CD62L(lo) CD4(+) T cells without associated IFN-γ production. Despite these Th2 features, sFliC immunization can enhance the development of protective Th1 immunity during subsequent Salmonella infection in an Ab-independent, T-cell-dependent manner. Salmonella infection in sFliC-immunized mice resulted in augmented Th1 responses, with greater bacterial clearance and increased numbers of IFN-γ-producing CD4(+) T cells, despite the early induction of Th2 features to sFliC. The augmented Th1 immunity after sFliC immunization was regulated by T-bet although T-bet is dispensable for primary responses to sFliC. These findings show that there can be flexibility in T-cell responses to some subunit vaccines. These vaccines may induce Th2-type immunity during primary immunization yet promote Th1-dependent responses during later infection. This suggests that designing Th1-inducing subunit vaccines may not always be necessary since this can occur naturally during subsequent infection.

Activated T regulatory (T(reg)) cells are potent suppressors that mediate immune tolerance. We investigated the relationship between activated T(reg) cells and the progression of human colon cancer. We designed a cross-sectional study of CD4(+) Foxp3(+) T cells from peripheral blood, primary tumor and nontumor colon tissue of 42 patients with colon cancer and correlated the percentages of different subgroups of T(reg) cells with colon cancer stage. The phenotypes, cytokine-release patterns and suppression ability of these T(reg) cells were analyzed. We found that T(reg) cells increased significantly in both peripheral blood and cancer tissue. In addition, the T(reg) cells expressed significantly lower levels of CCR7, CD62L and CD45RA in comparison to normal volunteers. Further dividing T(reg) cells into subgroups based on Foxp3 and CD45RA expression revealed that both activated T(reg) cells (Foxp3(hi) CD45RA(-)) and nonsuppressive T(reg) cells (Foxp3(lo) CD45RA(-)), but not resting T(reg) cells (Foxp3(low) CD45RA(+)), increased in the peripheral blood and cancer tissue of patients with colon cancer. Only the activated T(reg) cells expressed significantly higher levels of tumor necrosis factor receptor 2 and cytotoxic T-cell antigen-4. Activated T(reg) cells, however, secreted significantly lower levels of effector cytokines (interleukin-2, tumor necrosis factor-α and interferon-γ) than did resting T(reg) cells and nonsuppressive cells upon ex vivo stimulation. Activated, but not resting, T(reg) cells in cancer tissue correlated with tumor metastases. In summary, we confirmed that activated T(reg) cells are a distinct subgroup with effector memory phenotype and fully functional regulatory activity against human colorectal cancer immunity.

The use of hypocalcemic vitamin D analogs is an appealing strategy to exploit the immunomodulatory actions of active vitamin D in vivo while circumventing its calcemic side effects. The functional modulation of dendritic cells by these molecules is regarded as the key mechanism underlying their ability to regulate T cell reactivity. In this article, we demonstrate the capacity of the vitamin D analog, TX527, to target T cells directly. Microarray analysis of purified human CD3(+) T cells, cultured in the presence of TX527, revealed differential expression of genes involved in T cell activation, proliferation, differentiation, and migratory capacity. Accordingly, functional analysis showed a TX527-mediated suppression of the T cell proliferative capacity and activation status, accompanied by decreased expression of effector cytokines (IFN-γ, IL-4, and IL-17). Furthermore, TX527 triggered the emergence of CD4(+)CD25(high)CD127(low) regulatory T cells featuring elevated levels of IL-10, CTLA-4, and OX40 and the functional capacity to suppress activation and proliferation of effector T cells. Moreover, the vitamin D analog profoundly altered the homing receptor profile of T cells and their migration toward chemokine ligands. Remarkably, TX527 not only modulated skin-homing receptors as illustrated for the parent compound, but also reduced the expression of lymphoid organ-homing receptors (CD62L, CCR7, and CXCR4) and uniquely promoted surface expression of inflammatory homing receptors (CCR5, CXCR3, and CXCR6) on T cells. We conclude that TX527 directly affects human T cell function, thereby inhibiting effector T cell reactivity while inducing regulatory T cell characteristics, and imprints them with a specific homing signature favoring migration to sites of inflammation.

Alloantigen specific CD8+CD28- T suppressor (TS) cells differ from naturally occurring CD4+CD25+ T-regulatory (natural TR) cells not only by their phenotype but also by their mechanism of action. Natural TR have been extensively studied, leading to the identification of characteristic "molecular markers" such as Forkhead box P3 (FOXP3), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). We have investigated the expression of these genes in alloantigen specific TS and CD4+CD25+ T regulatory (TR) cells and found that they are expressed at levels similar to those observed in natural TR. Furthermore, similar to natural CD4+CD25+ TR, antigen-specific CD8+CD28-CD62L+ TS cells have more suppressive capacity than CD8+CD28-CD62L- TS cells. In spite of these similarities, natural TR are not antigen-specific and inhibit other T cells by T cell-to-T cell interaction, whereas TS are antigen-specific and exert their inhibitory function by interacting with antigen-presenting cells and render them tolerogenic to other T cells. The molecular characterization of TS cells may contribute to a better understanding of mechanisms involved in inhibition of immune responses in autoimmunity, transplantation, and chronic viral infection.

Plasmacytoid dendritic cells (PDC) are the natural type I IFN-producing cells that produce large amounts of IFN-alpha in response to viral stimulation. During attempts to isolate PDC from human PBMC, we observed that cross-linking a variety of cell surface receptors, including blood DC Ag (BDCA)-2, BDCA-4, CD4, or CD123 with Abs and immunobeads on PDC leads to inhibition of IFN-alpha production in response to HSV. To understand the mechanisms involved, a number of parameters were investigated. Cross-linking did not inhibit endocytosis of soluble Ag by PDC. Flow cytometry for annexin V and activated caspase-3 indicated that PDC are not undergoing apoptosis after receptor cross-linking. Cross-linking of CD123, but not the other receptors, caused the up-regulation of costimulatory molecules CD80 and CD86, as well as the down-regulation of CD62L, indicating PDC maturation. Thus, anti-CD123 Ab may be acting similar to the natural ligand, IL-3. Anti-phosphotyrosine Ab, as well as Ab to the IFN regulatory factor, IRF-7, was used in intracellular flow cytometry to elucidate the signaling pathways involved. Tyrosine phosphorylation occurred after cross-linking BDCA-2 and BDCA-4, but not CD4. Cross-linking did not affect IRF-7 levels in PDC, however, cross-linking BDCA-2, BDCA-4, and CD4, but not CD123, inhibited the ability of IRF-7 to translocate to the nucleus. Taken together, these results suggest that cross-linking BDCA-2, BDCA-4, and CD4 on PDC regulates IFN-alpha production at the level of IRF-7, while the decrease in IFN-alpha production after CD123 cross-linking is due to stimulation of the IL-3R and induction of PDC maturation.

Gammadelta T cells play an important role in innate immunity against infections; however, the regulation of these cells remains largely unknown. In the present study, we show that ESAT-6, an antigen of Mycobacterium tuberculosis, induces IFN-gamma secretion by human gammadelta T cells. In addition, ESAT-6 also induces the activation and proliferation of gammadelta T cells. Phenotypic analysis indicates that IFN-gamma-producing gammadelta T cells are mainly effector memory cells with the surface phenotype of CD45RA(-)CD62L(-)CCR7(-). These results were further confirmed by the fact that naive gammadelta T cells from cord blood did not produce IFN-gamma in response to ESAT-6. Further studies indicated that stimulation with ESAT-6 directly induced purified gammadelta T cells to produce IFN-gamma, independent of both antigen-presenting cells and CD4(+) T cells. Unexpectedly, depletion of CD4(+) T cells markedly enhanced IFN-gamma production by gammadelta T cells, indicating that CD4(+) T cells regulate the response of gammadelta T cells. Importantly, CD4(+)CD25(+) T regulatory (Treg) cells but not CD4(+)CD25(-) T cells significantly inhibited IFN-gamma production by gammadelta T cells. Taken together, these data demonstrate for the first time that Treg cells can play an important role in the regulation of immune responses of antigen-specific human memory gammadelta T cells.

Regulatory T cells that express the Foxp3 transcription factor play important roles in preventing autoimmune diseases. Although several studies have demonstrated that the lack of the forkhead DNA-binding domain of Foxp3 caused severe autoimmune disease in scurfy mutant mice, the other functional domains of Foxp3 are less well characterized. Here, we show that the deletion of glutamic acid (DeltaE250) in the leucine-zipper domain of Foxp3 causes a loss of hyporesponsiveness when compared with wild-type Foxp3 upon antigenic stimulation. CD4 T cells that ectopically express the glutamic acid mutant show significant losses of suppressor activity both in vitro and in vivo. We also demonstrate that regulation of both Th1- and Th2-type cytokine secretion in CD4 T cells that express wild-type Foxp3 is significantly altered by the deletion of glutamic acid. Defects are also observed in the expression of adhesion molecules, such as l-selectin (CD62L) and CD103, suggesting an important role of glutamic acid in the migratory behavior of regulatory T cells. Finally, this mutation reduces transcriptional repressor activity and impairs the homodimerization of Foxp3. Taken together, our results provide insight into the mechanism that controls autoimmune diseases via the deletion of this single glutamic acid residue in the leucine-zipper domain of Foxp3.

Granulocyte colony-stimulating factor (G-CSF) may affect T-cell homeostasis by multiple mechanisms, inducing polarization of cytokine secretion, inhibition of T-cell proliferation, and enhancement of T-cell apoptosis. We analyzed the production of interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1) by T cells from healthy volunteer donors treated with recombinant human G-CSF. Highly purified CD4(+) T cells obtained before and after G-CSF administration (pre-G and post-G, respectively) were activated using the allogeneic mixed leukocyte reaction. Post-G CD4(+) T cells produced high levels of IL-10 but undetectable levels of IL-2 and IL-4, whereas the level of TGF-beta1 release was comparable to that of pre-G CD4(+) T cells. Notably, post-G CD4(+) T cells proliferated poorly in response to alloantigens and to recall antigens and suppressed the proliferation of autologous CD4(+) T cells in a cell contact-independent and an antigen-nonspecific manner. TGF-beta1 and IL-10 were not dispensable for post-G CD4(+) T cells to mediate suppression, as shown by neutralization studies. Compared with pre-G CD4(+) T cells, alloantigen-activated post-G CD4(+) T cells preferentially expressed markers associated with memory T cells, in conjunction with reduced levels of CD28 and CD62L. Collectively, these data demonstrate that CD4(+) T cells exposed to G-CSF in vivo acquire the properties of T regulatory (Tr) cells once triggered in vitro through the T-cell receptor, including a peculiar cytokine production profile (IL-10(++)TGF-beta1(+)IL-2(low/-)IL-4(low/-)), an intrinsic low proliferative capacity, and a contact-independent suppression of antigen-driven proliferation. Tr cells generated ex vivo after exposure to G-CSF might be clinically relevant for transplantation medicine and for the treatment of human immune-mediated diseases.

To more closely understand the mechanisms of how BCG vaccination confers immunity would help to rationally design improved tuberculosis vaccines that are urgently required. Given the established central role of CD4 T cells in BCG induced immunity, we sought to characterise the generation of memory CD4 T cell responses to BCG vaccination and M. bovis infection in a murine challenge model. We demonstrate that a single systemic BCG vaccination induces distinct systemic and mucosal populations of T effector memory (T(EM)) cells in vaccinated mice. These CD4+CD44(hi)CD62L(lo)CD27⁻ T cells concomitantly produce IFN-γ and TNF-α, or IFN-γ, IL-2 and TNF-α and have a higher cytokine median fluorescence intensity MFI or 'quality of response' than single cytokine producing cells. These cells are maintained for long periods (>16 months) in BCG protected mice, maintaining a vaccine-specific functionality. Following virulent mycobacterial challenge, these cells underwent significant expansion in the lungs and are, therefore, strongly associated with protection against M. bovis challenge. Our data demonstrate that a persistent mucosal population of T(EM) cells can be induced by parenteral immunization, a feature only previously associated with mucosal immunization routes; and that these multifunctional T(EM) cells are strongly associated with protection. We propose that these cells mediate protective immunity, and that vaccines designed to increase the number of relevant antigen-specific T(EM) in the lung may represent a new generation of TB vaccines.

Dendritic cells (DC) play a major role in the pathogenesis of graft-vs-host disease (GvHD). Directed modification of surface molecules on DC that provide instructive signals for T cells may create a tolerogenic DC phenotype that affects GvHD severity. To investigate the impact of the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) on in vivo migratory capacities, tolerogenic function, and B7 superfamily surface expression on DC following allogeneic hematopoietic cell transplantation (aHCT), we generated a platform for magnetic resonance imaging and bioluminescence imaging based cell trafficking studies. Luciferase transgenic DC were labeled with superparamagnetic iron oxide nanoparticles bound to a murine IgG Ab that allowed for Fc-gammaR-mediated endocytosis. Locally injected luc(+) DC could be tracked within their anatomical context by bioluminescence imaging and magnetic resonance imaging after aHCT, based on stable intracellular localization of superparamagnetic iron oxide-IgG complexes. RAPA preconditioned DC (DC-R) displayed reduced expression of MHC class II, B7-1 (CD80), and B7-2 (CD86) but not B7-H4 whose ligation of T cells has a profound inhibitory effect on their proliferation and cytokine secretion. DC-R of recipient genotype reduced GvHD severity that is compatible with their tolerogenic phenotype. CCR5, CCR7, and CD62L expression was not affected by mTOR inhibition, which allowed for DC-R in vivo trafficking to secondary lymphoid compartments where immunregulation is required. This study is the first to delineate the impact of RAPA on DC migration and tolerogenic function after aHCT. Modification of the DC phenotype by mTOR inhibition may have therapeutic potential in an attempt to reduce GvHD following aHCT.

CD8(+) T cells are likely to play an important role in host defense against Salmonella enterica serovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-gamma) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8(+) T cells in humans. To this end, we determined the TCR beta chain (Vbeta) usage of CD8(+) T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR Vbeta usage varied among volunteers, we identified oligoclonal Vbeta subset expansions in individual volunteers (Vbeta 2, 5.1, 8, 17, and 22 in volunteer 1; Vbeta 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and Vbeta 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN-gamma secretion assays on Vbeta sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7(-) CD27(-) CD45RO(+) CD62L(-)) and coexpressed gut homing molecules (e.g., high levels of integrin alpha4beta7, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR Vbeta families. Moreover, these serovar Typhi-specific CD8(+) T cells bearing defined Vbeta specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential.

CD4(+) memory/effector T cells play a central role in orchestrating the rapid and robust immune responses upon re-encounter with specific Ags. However, the immunologic mechanism(s) underlying these responses are still not fully understood. To investigate this, we generated an allergen (major house dust mite allergen, Blo t 5)-specific murine Th2 cell line that secreted IL-4, IL-5, IL-10, and IL-13, but not IL-9 or TNF-α, upon activation by the cognate Ag. These cells also exhibited CD44(high)CD62L(-) and CD127(+) (IL-7Rα(+)) phenotypes, which are characteristics of memory/effector T cells. Experiments involving adoptive transfer of this Th2 cell line in mice, followed by three intranasal challenges with Blo t 5, induced a dexamethasone-sensitive eosinophilic airway inflammation. This was accompanied by elevation of Th2 cytokines and CC- and CXC-motif chemokines, as well as recruitment of lymphocytes and polymorphic mononuclear cells into the lungs. Moreover, Blo t 5-specific IgE was detected 4 d after the last intranasal challenge, whereas elevation of Blo t 5-specific IgG1 was found at week two. Finally, pulmonary delivery of the pVAX-IL-35 DNA construct effectively downregulated Blo t 5-specific allergic airway inflammation, and i.m. injection of pVAX-IL-35 led to long-lasting suppression of circulating Blo t 5-specific and total IgE. This model provides a robust research tool to elucidate the immunopathogenic role of memory/effector Th2 cells in allergic airway inflammation. Our results suggested that IL-35 could be a potential therapeutic target for allergic asthma through its attenuating effects on allergen-specific CD4(+) memory/effector Th2 cell-mediated airway inflammation.

Although human immunodeficiency virus (HIV)-infected subjects without AIDS have a high frequency of HIV-specific CD8 T lymphocytes, cellular immunity is unable to control infection. Freshly isolated lymphocytes often do not lyse HIV-infected targets in 4-h cytotoxicity assays. A large fraction of circulating CD8 T cells from HIV-infected donors down-modulate CD3zeta, the signaling component of the T-cell receptor complex, which is reexpressed in vitro coincident with the return of cytotoxic function. To investigate further the link between CD3zeta down-modulation and possible CD8 T-cell functional defects, we used flow cytometry to characterize further the properties of the CD3zeta-down-modulated subset. HIV-specific CD8 T cells, identified by tetramer staining, are CD3zeta(-). CD8 T cells with down-modulated CD3zeta also do not express the key costimulatory receptor CD28 and have the cell surface phenotype of activated or memory T cells (HLA-DR(+) CD62L(-)). After T-cell activation, CD3zeta-down-modulated cells express the activation marker CD69 but not the high-affinity interleukin 2 (IL-2) receptor alpha-chain CD25 and produce gamma interferon but not IL-2. Therefore HIV-specific CD8 T cells have down-modulated key signaling molecules for T-cell activation and costimulation and require exogenous cytokine stimulation. The typical impairment of HIV-specific CD4 T helper cells, which would normally provide specific CD8 T-cell stimulation, means that in vivo CTL function in vivo is compromised in most HIV-infected individuals. In AIDS patients, the functional defect is more severe, since CD3zeta is not reexpressed even after IL-2 exposure.

Infection with the protozoan parasite Trypanosoma cruzi is a major cause of morbidity and mortality in Central and South America. Control of acute experimental infection with T. cruzi is dependent on a robust T cell and type 1 cytokine response. However, little evidence exists demonstrating the development and persistence of a potent antiparasite T cell memory response, and there has been much speculation that the majority of the immune response to T. cruzi infection is not directed against the parasite. In this study, we used an experimental mouse model of T. cruzi infection to test both the Ag specificity and the functional and phenotypic characteristics of the responding T cell population. We observed a vigorous antiparasite response from both CD4(+) and CD8(+) T cells that was maintained in the face of persistent infection. T cells from infected mice also proliferated in response to re-exposure to Ag, and CD8(+) T cells underwent spontaneous proliferation when transferred to naive congenic mice, both characteristic of central memory T cells. Interestingly, T cells from infected mice showed significant down-regulation of CD62L, a characteristic associated with an effector memory phenotype. These results suggest that T cells maintained in mice with chronic T. cruzi infection are fully functional memory cells that cannot be easily categorized in the current central/effector memory paradigm.

MRL/MpJ-Fas(lpr/lpr)/J (MRL(lpr)) mice develop lupus-like disease manifestations in an IL-21-dependent manner. IL-21 is a pleiotropic cytokine that can influence the activation, differentiation, and expansion of B and T cell effector subsets. Notably, autoreactive CD4(+) T and B cells spontaneously accumulate in MRL(lpr) mice and mediate disease pathogenesis. We sought to identify the particular lymphocyte effector subsets regulated by IL-21 in the context of systemic autoimmunity and, thus, generated MRL(lpr) mice deficient in IL-21R (MRL(lpr).IL-21R(-/-)). Lymphadenopathy and splenomegaly, which are characteristic traits of the MRL(lpr) model were significantly reduced in the absence of IL-21R, suggesting that immune activation was likewise decreased. Indeed, spontaneous germinal center formation and plasma cell accumulation were absent in IL-21R-deficient MRL(lpr) mice. Correspondingly, we observed a significant reduction in autoantibody titers. Activated CD4(+) CD44(+) CD62L(lo) T cells also failed to accumulate, and CD4(+) Th cell differentiation was impaired, as evidenced by a significant reduction in CD4(+) T cells that produced the pronephritogenic cytokine IFN-γ. T extrafollicular helper cells are a recently described subset of activated CD4(+) T cells that function as the primary inducers of autoantibody production in MRL(lpr) mice. Importantly, we demonstrated that T extrafollicular helper cells are dependent on IL-21R for their generation. Together, our data highlighted the novel observation that IL-21 is a critical regulator of multiple pathogenic B and T cell effector subsets in MRL(lpr) mice.