Yanling Xiao

CD27 Promotes Survival of Activated T Cells and Complements CD28 in Generation and Establishment of the Effector T Cell Pool

CD27, like CD28, acts in concert with the T cell receptor to support T cell expansion. Using CD27−/− mice, we have shown earlier that CD27 determines the magnitude of primary and memory T cell responses to influenza virus. Here, we have examined the relative contributions of CD27 and CD28 to generation of the virus-specific effector T cell pool and its establishment at the site of infection (the lung), using CD27−/−, CD28−/−, and CD27/CD28−/− mice. We find that primary and memory CD8+ T cell responses to influenza virus are dependent on the collective contribution of both receptors. In the primary response, CD27 and CD28 impact to a similar extent on expansion of virus-specific T cells in draining lymph nodes. CD27 is the principle determinant for accumulation of virus-specific T cells in the lung because it can sustain this response in CD28−/− mice. Unlike CD28, CD27 does not affect cell cycle activity, but promotes survival of activated T cells throughout successive rounds of division at the site of priming and may do so at the site of infection as well. CD27 was found to rescue CD28−/− T cells from death at the onset of division, explaining its capacity to support a T cell response in absence of CD28.

During Viral Infection of the Respiratory Tract, CD27, 4-1BB, and OX40 Collectively Determine Formation of CD8+ Memory T Cells and Their Capacity for Secondary Expansion

Independent studies have shown that CD27, 4-1BB, and OX40 can all promote survival of activated CD8+ T cells. We have therefore compared their impact on CD8+ memory T cell formation and responsiveness within one, physiologically relevant model system. Recombinant mice, selectively lacking input of one or two receptors, were challenged intranasally with influenza virus, and the immunodominant virus-specific CD8+ T cell response was quantified at priming and effector sites. Upon primary infection, CD27 and (to a lesser extent) 4-1BB made nonredundant contributions to accumulation of CD8+ virus-specific T cells in draining lymph nodes and lung, while OX40 had no effect. Interestingly though, in the memory response, accumulation of virus-specific CD8+ T cells in spleen and lung critically depended on all three receptor systems. This was explained by two observations: 1) CD27, 4-1BB, and OX40 were collectively responsible for generation of the same memory CD8+ T cell pool; 2) CD27, 4-1BB, and OX40 collectively determined the extent of secondary expansion, as shown by adoptive transfers with standardized numbers of memory cells. Surprisingly, wild-type CD8+ memory T cells expanded normally in primed OX40 ligand- or 4-1BB ligand-deficient mice. However, when wild-type memory cells were generated in OX40 ligand- or 4-1BB ligand-deficient mice, their secondary expansion was impaired. This provides the novel concept that stimulation of CD8+ T cells by OX40 and 4-1BB ligand during priming imprints into them the capacity for secondary expansion. Our data argue that ligand on dendritic cells and/or B cells may be critical for this.

Expression of the murine CD27 ligand CD70 in vitro and in vivo

The interaction between TNFR family member CD27 and its ligand CD70 promotes lymphocyte expansion and effector cell formation. In humans, control of CD27 function is partly regulated by the restricted expression of CD70. We used newly developed mAbs to characterize murine (m) CD70 expression in vitro and in vivo. On resting lymphocytes and immature dendritic cells (DC), mCD70 is absent. In vitro, Ag receptor triggering induced mCD70 mRNA in T cells, but cell surface protein expression was very low. Activated B cells synthesized much higher levels of mCD70 mRNA than activated T cells and clearly expressed mCD70 at the cell surface. mCD70 cell surface expression could also be induced on the DC line D1 and on in vitro-generated murine DC upon maturation. In lymphoid organs of naive mice, virtually no mCD70-expressing cells were found, with exception of cells in the thymic medulla, which may be epithelial in origin. However, after intranasal infection with influenza virus, lung-infiltrating T cells and T and B cells in draining lymph nodes expressed mCD70 according to immunohistology. In such activated lymphocytes, mCD70 protein is largely retained intracellularly. Plasma membrane expression of mCD70 was only detectable by flow cytometry on a small proportion of lung-infiltrating T cells and peaked at the height of the primary response. Thus, expression of CD70 in the mouse is highly regulated at the transcriptional and posttranslational level. This most likely serves to limit excessive effector cell formation after antigenic stimulation.

Expression of Costimulatory Ligand CD70 on Steady-State Dendritic Cells Breaks CD8+ T Cell Tolerance and Permits Effective Immunity

Steady-state dendritic cells (DCs) maintain peripheral T cell tolerance, whereas mature DCs generate immunity. CD70 is a costimulatory ligand acquired upon DC maturation. To determine its impact on T cell fate, we have generated mice that constitutively express CD70 in conventional DCs (cDCs). In these mice, naive CD4+ and CD8+ T cells spontaneously convert into effector cells. Administration of peptide without adjuvant, which is ordinarily tolerogenic, elicited tumor-eradicating CD8+ T cell responses and robust CD4+ T cell-independent memory. CD70 was also constitutively expressed in cDCs that inducibly present viral epitopes. In this case, tolerance induction was prevented as well. The antigen-presenting DCs generated protective immunity to virus infection and broke a pre-existing state of CD8+ T cell tolerance. Thus, the sole expression of CD70 by otherwise immature cDCs sufficed to convert CD8+ T cell tolerance into immunity, defining the importance of CD27-CD70 interactions at the interface between T cell and DC.

CD27 Is Acquired by Primed B Cells at the Centroblast Stage and Promotes Germinal Center Formation

Studies on human B cells have featured CD27 as a marker and mediator of the B cell response. We have studied CD27 expression and function on B cells in the mouse. We find that B cells acquire CD27 at the centroblast stage and lose it progressively upon further differentiation. It is not a marker for somatically mutated B cells and is present at very low frequency on memory B cells. Enrichment of CD27 among centroblasts and the presence of its ligand CD70 on occasional T and B cells in or near germinal centers (GCs) suggested a role for CD27/CD70 interactions in clonal B cell expansion. Accordingly, GC formation in response to influenza virus infection was delayed in CD27 knockout mice. CD27 deficiency did not affect somatic hypermutation or serum levels of virus-specific IgM, IgG, and IgA attained in primary and recall responses. Adoptive transfer of T and B cells into CD27/CD28−/− mice revealed that CD27 promotes GC formation and consequent IgG production by two distinct mechanisms. Stimulation of CD27 on B cells by CD28+ Th cells accelerates GC formation, most likely by promoting centroblast expansion. In addition, CD27 on T cells can partially substitute for CD28 in supporting GC formation.

CD27 sustains survival of CTLs in virus-infected nonlymphoid tissue in mice by inducing autocrine IL-2 production

Immunity to infections relies on clonal expansion of CD8+ T cells, their maintenance as effector CTLs, and their selection into a memory population. These processes rely on delivery of survival signals to activated CD8+ T cells. We here reveal the mechanism by which costimulatory CD27-CD70 interactions sustain survival of CD8+ effector T cells in infected tissue. By unbiased genome-wide gene expression analysis, we identified the Il2 gene as the most prominent CD27 target gene in murine CD8+ T cells. In vitro, CD27 directed IL-2 expression and promoted clonal expansion of primed CD8+ T cells exclusively by IL-2-dependent survival signaling. In mice intranasally infected with influenza virus, Cd27-/- CD8+ effector T cells displayed reduced IL-2 production, accompanied by impaired accumulation in lymphoid organs and in the lungs, which constitute the tissue effector site. Reconstitution of Cd27-/- CD8+ T cells with the IL2 gene restored their accumulation to wild-type levels in the lungs, but it did not rescue their accumulation in lymphoid organs. Competition experiments showed that the IL-2 produced under the control of CD27 supported effector CD8+ T cell survival in the lungs in an autocrine manner. We conclude that CD27 signaling directs the IL-2 production that is reportedly essential to sustain survival of virus-specific CTLs in nonlymphoid tissue.

Epithelial and dendritic cells in the thymic medulla promote CD4+Foxp3+ regulatory T cell development via the CD27-CD70 pathway

CD27–CD70 signals are required for optimal development of natural regulatory T cells from the thymus.

CD27 instructs CD4+ T cells to provide help for the memory CD8+ T cell response after protein immunization.

For optimal quality, memory CD8+ T cells require CD4+ T cell help. We have examined whether CD4+ T cells require CD27 to deliver this help, in a model of intranasal OVA protein immunization. CD27 deficiency reduced the capacity of CD4+ T cells to support Ag-specific CD8+ T cell accumulation at the tissue site after primary and secondary immunization. CD27-dependent CD4+ T cell help for the memory CD8+ T cell response was delivered during priming. It did not detectably affect formation of CD8+ memory T cells, but promoted their secondary expansion. CD27 improved survival of primed CD4+ T cells, but its contribution to the memory CD8+ T cell response relied on altered CD4+ T cell quality rather than quantity. CD27 induced a Th1-diagnostic gene expression profile in CD4+ T cells, which included the membrane molecule MS4A4B. Accordingly, CD27 increased the frequency of IFN-γ- and IL-2-producing CD4+ T cells. It did not affect CD40L expression. Strikingly, MS4A4B was also identified as a unique marker of CD8+ memory T cells that had received CD27-proficient CD4+ T cell help during the primary response. This apparent imprinting effect suggests a role for MS4A4B as a downstream effector in CD27-dependent help for CD8+ T cell memory.

Costimulatory ligand CD70 allows induction of CD8+ T-cell immunity by immature dendritic cells in a vaccination setting

The use of dendritic cells (DCs) as anticancer vaccines holds promise for therapy but requires optimization. We have explored the potential of costimulatory ligand CD70 to boost the capacity of DCs to evoke effective CD8(+) T-cell immunity. We show that immature conventional DCs, when endowed with CD70 expression by transgenesis, are converted from a tolerogenic state into an immunogenic state. Adoptively transferred CD70-expressing immature DCs could prime CD8(+) T cells, by CD27, to become tumor-eradicating cytolytic effectors and memory cells with a capacity for robust secondary expansion. The CD8(+) T-cell response, including memory programming, was independent of CD4(+) T-cell help, because the transferred immature DCs were loaded with major histocompatibility complex class I-restricted peptide only. Without CD70 expression, the DCs generated abortive clonal expansion, dysfunctional antitumor responses, and no CD8(+) T-cell memory. CD70-expressing CD8(+) DCs were the primary subset responsible for CD8(+) T-cell priming and performed comparably to fully matured DCs. These data highlight the importance of CD27/CD70 interactions at the T-cell/DC interface and indicate that CD70 should be considered in the design of DC vaccination strategies.

Dexamethasone treatment during the expansion phase maintains stemness of bone marrow mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) can form various mesodermal tissues when grown under appropriate conditions. Dexamethasone (Dex) is regularly used to stimulate the osteogenic potential of hMSCs and it has recently been reported to increase the cell expansion rate. In this study we have investigated the effect of low‐dose Dex treatment (10−8 M) on the multipotency of expanded hMSCs, using histological, biochemical and molecular biological techniques. Early passage (P2‐3) and late passage (P6) cells were positive (>90%) for mesenchymal adhesion cell markers (CD105/CD29/CD44/CD166/CD90) and negative (<10%) for haematopoietic markers (CD34/CD45/CD14). Dex did not change the overall expression pattern of these cell surface markers. Expanded hMSCs gave rise to specialized cell lineages when grown in differentiation‐promoting medium. Depending on the donor, Dex treatment improved the potency for osteogenic, adipogenic and chondrogenic differentiation of expanded hMSCs. Dex also prevented the loss of proliferative potential of hMSCs upon sequential passaging and the loss of the typical hMSCs surface phenotype. hMSCs gene expression analysis showed that low‐dose Dex negatively regulated transcription of genes correlated with apoptosis and differentiation, and positively regulated genes associated with cell proliferation. In conclusion, the collective data argue that low‐dose Dex preserves the stemness of hMSCs during repeated passaging, as indicated by the maintenance of the stem cell phenotype, proliferative capacity and multi‐lineage differentiation potential. Copyright