Maria Fernanda Pascutti

IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells

Chronic lymphocytic leukemia (CLL) cells multiply in secondary lymphoid tissue, but the mechanisms leading to their proliferation are still uncertain. In addition to B-cell receptor (BCR)-triggered signals, other microenvironmental factors might well be involved. In proliferation centers, leukemic B cells are in close contact with CD4(+)CD40L(+) T cells. Therefore, we here dissected the signals provided by autologous activated T cells (Tact) to CLL cells. Although the gene expression profile induced by Tact was highly similar to that induced by sole CD40 signaling, an obvious difference was that Tact induced proliferation of CLL cells. We determined that stimulation with only CD40L+IL-21 was sufficient to induce robust proliferation in CLL cells. We then defined an interleukin (IL)-21-induced gene signature in CLL, containing components of Janus kinase/signal transducer and activator of transcription and apoptosis pathways, and this signature could be detected in lymph node (LN) samples from patients. Finally, we could detect IL-21 RNA and protein in LN, and IL-21 production ex vivo by LN CD4(+)CXCR5(+) follicular helper T cells. These results indicate that in addition to BCR signaling, activated T cells might contribute to CLL cell proliferation via CD40 and IL-21. Targeting these signaling pathways might offer new venues for treatment of CLL.

CMV-specific CD8+ T-cell function is not impaired in chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL), CD8(+) T cells exhibit features of exhaustion and impaired functionality. Yet, reactivations of latent viruses such as cytomegalovirus (CMV) are uncommon in untreated CLL, suggesting that antiviral responses are uncompromised. We analyzed phenotypical and functional characteristics of CMV-specific CD8(+) T cells in CLL patients in comparison with age-matched healthy controls (HCs). Despite increased expression of the inhibitory receptors PD1, CD160, and CD244 on total CD8(+) T cells in CLL, expression levels of these markers were decreased on CMV-tetramer(+)CD8(+) T cells. Second, cytokine production upon stimulation with both phorbol 12-myristate 13-acetate/ionomycin and CMV-peptide-loaded antigen-presenting cells was intact in CMV-tetramer(+)CD8(+) T cells. Third, CMV-tetramer(+)CD8(+) T cells of CLL patients and HCs were equally effective in killing CMV-peptide-loaded target cells. Finally, quantitative imaging flow cytometry revealed that the proportion of CD8(+) T cells forming immunologic synapses with CMV-peptide-loaded B cells was intact. In conclusion, despite evidence for global T-cell dysfunction in CLL, we show here that CLL-derived CMV-specific CD8(+) T cells display lower expression of exhaustion markers and are functionally intact. These data indicate that the changes in the T-cell compartment in CLL may be more heterogeneous than presently assumed.

Pro-Apoptotic Protein Noxa Regulates Memory T Cell Population Size and Protects against Lethal Immunopathology

Memory T cells form a highly specific defense layer against reinfection with previously encountered pathogens. In addition, memory T cells provide protection against pathogens that are similar, but not identical to the original infectious agent. This is because each T cell response harbors multiple clones with slightly different affinities, thereby creating T cell memory with a certain degree of diversity. Currently, the mechanisms that control size, diversity, and cross-reactivity of the memory T cell pool are incompletely defined. Previously, we established a role for apoptosis, mediated by the BH3-only protein Noxa, in controlling diversity of the effector T cell population. This function might positively or negatively impact T cell memory in terms of function, pool size, and cross-reactivity during recall responses. Therefore, we investigated the role of Noxa in T cell memory during acute and chronic infections. Upon influenza infection, Noxa−/− mice generate a memory compartment of increased size and clonal diversity. Reinfection resulted in an increased recall response, whereas cross-reactive responses were impaired. Chronic infection of Noxa−/− mice with mouse CMV resulted in enhanced memory cell inflation, but no obvious pathology. In contrast, in a model of continuous, high-level T cell activation, reduced apoptosis of activated T cells rapidly led to severe organ pathology and premature death in Noxa-deficient mice. These results establish Noxa as an important regulator of the number of memory cells formed during infection. Chronic immune activation in the absence of Noxa leads to excessive accumulation of primed cells, which may result in severe pathology.

Enhanced CD8 T Cell Responses through GITR-Mediated Costimulation Resolve Chronic Viral Infection

Chronic infections are characterized by the inability to eliminate the persisting pathogen and often associated with functional impairment of virus-specific T-cell responses. Costimulation through Glucocorticoid-induced TNFR-related protein (GITR) can increase survival and function of effector T cells. Here, we report that constitutive expression of GITR-ligand (GITRL) confers protection against chronic lymphocytic choriomeningitis virus (LCMV) infection, accelerating recovery without increasing pathology. Rapid viral clearance in GITRL transgenic mice coincided with increased numbers of poly-functional, virus-specific effector CD8+ T cells that expressed more T-bet and reduced levels of the rheostat marker PD-1. GITR triggering also boosted the helper function of virus-specific CD4 T cells already early in the infection, as was evidenced by increased IL-2 and IFNγ production, and more expression of CD40L and T-bet. Importantly, CD4-depletion experiments revealed that the expanded pool of virus-specific effector CD8 T cells and the ensuing viral clearance in LCMV-infected GITRL tg mice was entirely dependent on CD4 T cells. We found no major differences for NK cell and regulatory T cell responses, whereas the humoral response to the virus was increased in GITRL tg mice, but only in the late phase of the infection when the virus was almost eradicated. Based on these findings, we conclude that enhanced GITR-triggering mediates its protective, anti-viral effect on the CD8 T cell compartment by boosting CD4 T cell help. As such, increasing costimulation through GITR may be an attractive strategy to increase anti-viral CTL responses without exacerbating pathology, in particular to persistent viruses such as HIV and HCV.

Quantitative and Qualitative Analysis of Bone Marrow CD8+ T Cells from Different Bones Uncovers a Major Contribution of the Bone Marrow in the Vertebrae

Bone marrow (BM) plays an important role in the long-term maintenance of memory T cells. Yet, BM is found in numerous bones throughout the body, which are not equal in structure, as they differ in their ratio of cortical and trabecular bone. This implies that BM cells within different bones are subjected to different microenvironments, possibly leading to differences in their frequencies and function. To address this, we examined BM from murine tibia, femur, pelvis, sternum, radius, humerus, calvarium, and the vertebrae and analyzed the presence of effector memory (TEM), central memory (TCM), and naïve (TNV) CD8+ T cells. During steady-state conditions, the frequency of the total CD8+ T cell population was comparable between all bones. Interestingly, most CD8+ T cells were located in the vertebrae, as it contained the highest amount of BM cells. Furthermore, the frequencies of TEM, TCM, and TNV cells were similar between all bones, with a majority of TNV cells. Additionally, CD8+ T cells collected from different bones similarly expressed the key survival receptors IL-7Rα and IL-15Rβ. We also examined BM for memory CD8+ T cells with a tissue-resident memory phenotype and observed that approximately half of all TEM cells expressed the retention marker CD69. Remarkably, in the memory phase of acute infection with the lymphocytic choriomeningitis virus (LCMV), we found a massive compositional change in the BM CD8+ T cell population, as the TEM cells became the dominant subset at the cost of TNV cells. Analysis of Ki-67 expression established that these TEM cells were in a quiescent state. Finally, we detected higher frequencies of LCMV-specific CD8+ T cells in BM compared to spleen and found that BM in its entirety contained fivefold more LCMV-specific CD8+ T cells. In conclusion, although infection with LCMV caused a dramatic change in the BM CD8+ T cell population, this did not result in noticeable differences between BM collected from different bones. Our findings suggest that in respect to CD8+ T cells, BM harvested from a single bone is a fair reflection of the rest of the BM present in the murine body.

Impact of Viral Infections on Hematopoiesis: From Beneficial to Detrimental Effects on Bone Marrow Output

The ability of the bone marrow (BM) to generate copious amounts of blood cells required on a daily basis depends on a highly orchestrated process of proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). This process can be rapidly adapted under stress conditions, such as infections, to meet the specific cellular needs of the immune response and the ensuing physiological changes. This requires a tight regulation in order to prevent either hematopoietic failure or transformation. Although adaptation to bacterial infections or systemic inflammation has been studied and reviewed in depth, specific alterations of hematopoiesis to viral infections have received less attention so far. Viruses constantly pose a significant health risk and demand an adequate, balanced response from our immune system, which also affects the BM. In fact, both the virus itself and the ensuing immune response can have a tremendous impact on the hematopoietic process. On one hand, this can be beneficial: it helps to boost the cellular response of the body to resolve the viral infection. But on the other hand, when the virus and the resulting antiviral response persist, the inflammatory feedback to the hematopoietic system will become chronic, which can be detrimental for a balanced BM output. Chronic viral infections frequently have clinical manifestations at the level of blood cell formation, and we summarize which viruses can lead to BM pathologies, like aplastic anemia, pancytopenia, hemophagocytic lymphohistiocytosis, lymphoproliferative disorders, and malignancies. Regarding the underlying mechanisms, we address specific effects of acute and chronic viral infections on blood cell production. As such, we distinguish four different levels in which this can occur: (1) direct viral infection of HSPCs, (2) viral recognition by HSPCs, (3) indirect effects on HSPCs by inflammatory mediators, and (4) the role of the BM microenvironment on hematopoiesis upon virus infection. In conclusion, this review provides a comprehensive overview on how viral infections can affect the formation of new blood cells, aiming to advance our understanding of the underlying cellular and molecular mechanisms to improve the treatment of BM failure in patients.

Constitutive GITR Activation Reduces Atherosclerosis by Promoting Regulatory CD4+ T-Cell Responses—Brief Report

Objective—Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is expressed on CD4+ effector memory T cells and regulatory T cells; however, its role on these functionally opposing cell types in atherosclerosis is not fully understood. Approach and Results—Low-density lipoprotein receptor–deficient mice (Ldlr−/−) were lethally irradiated and reconstituted with either bone marrow from B-cell–restricted Gitrl transgenic mice or from wild-type controls and fed a high-cholesterol diet for 11 weeks. Chimeric Ldlr−/− Gitrltg mice showed a profound increase in both CD4+ effector memory T cells and regulatory T cells in secondary lymphoid organs. Additionally, the number of regulatory T cells was significantly enhanced in the thymus and aorta of these mice along with increased Gitrl and Il-2 transcript levels. Atherosclerotic lesions of Ldlr−/− Gitrltg chimeras contained more total CD3+ T cells as well as Foxp3+ regulatory T cells overall, leading to significantly less severe atherosclerosis. Conclusions—These data indicate that continuous GITR stimulation through B cell Gitrl acts protective in a mouse model of atherosclerosis by regulating the balance between regulatory and effector memory CD4+ T cells.

Memory CD8+ T cells support the maintenance of hematopoietic stem cells in the bone marrow.

Following viral infections, considerable numbers of virus-specific memory T cells accumulate in the bone marrow (BM).[1][1] This organ is rich in interleukin-15, which is required for the long-term maintenance of memory T cells.[2][2] Whether this is driven by local homeostatic proliferation or by