Diabetic nephropathy (DN) is a common complication in patients with diabetes, and most of them need renal replacement therapy such as haemodialysis (HD). These patients have a high tendency to develop infections and exhibit anomalies in the immune system. The objective of this study was to assess the expression of activation-related markers on T cells, as well as to quantify inflammatory cytokines, before and after a single HD session in DN patients. The study involved DN patients under HD treatment who signed an informed consent form. Blood samples before and after one HD session were collected, to analyse the expression of CD25, CD69 and CD71 in T cells. We also quantified IL-12p70, IL-8, IL-10, IL-1β, TNF-α and IL-6 in serum samples using the cytometric bead array technique. After the HD session, there was an increase in the CD4/CD8 ratio due to significant alterations in both subsets. The relative percentage of CD25+ cells and CD8+ CD25+ increased significantly after the HD session, while the relative percentage of CD69 T cells decreased. There was a significant decrease in the CD25 mean fluorescence intensity values for CD4+ T, as well as in the case of CD71 in T cells after the HD session. Regarding cytokine synthesis, we found a significant increase in IL-10 and IL-6 and a decrease in IL-8 after HD session. This study showed that a HD session in DN patients affects the T cell activation status in the two major subpopulations and differentially modulates the production of inflammatory cytokines.

X-linked severe combined immunodeficiency disease (SCID) is caused by mutations in the interleukin (IL)-2 receptor γ (IL2RG) gene and patients usually present with a TBNK SCID phenotype. Nevertheless, a minority of these patients present with a TBNK phenotype, similar to the IL-7R-deficient patients. We report a patient with a novel missense p.Glu297Gly mutation in the IL2RG gene presenting with a leaky TBNK SCID with delayed onset, moderate susceptibility to infections, and nodular regenerative hyperplasia. He presents with preserved STAT5 tyrosine phosphorylation in response to IL-15 stimulation but not in response to IL-2 and IL-7, resulting in the NK phenotype.

Haematopoiesis is a developmental process that ensures the generation of all blood cell lineages throughout life. As a consequence, this is a highly complex and dynamic developmental cascade subject to tight regulatory mechanisms. Thus, the study of novel molecular signals is critical to further understand how haematopoiesis operates to ensure the balance between cell lineage commitment, cell homeostasis and efficient haematopoietic responses to insults and disturbances. The tyrosine kinase RET is the receptor for the GDNF (glial cell line-derived neurotrophic factor) neurotrophic factor family (GDNF family ligands – GFLs). Productive RET signalling controls the development and maintenance of the enteric nervous system, kidneys and spermatogenesis. Interestingly, Ret expression was detected in haematopoietic cells and lymphoid organs and RET signalling was shown to regulate enteric lymphoid organogenesis. However the role of RET in haematopoiesis remains completely unexplored. Haematopoietic stem cells (HSCs) are at the onset of the developmental cascade that generate all blood cells, thus we initially investigated the role of RET in HSC function and how modulation of RET signalling can be used to control HSC responses. Finally, we investigated the role of RET in late stages of haematopoietic cell precursor differentiation into the T cell lineage. We found that the tyrosine kinase RET is critical to HSC survival and function. HSCs express RET signalling molecules and HSCs microenvironment provides RET ligands. Moreover Ret ablation leads to reduced HSC numbers and recruitment of quiescent cells into proliferation. Although RET null progenitors have normal differentiation potential, they exhibit impaired in vivo stress response and reconstitution potential. Remarkably RET downstream signalling results in p38/MAP kinase phosphorylation and CREB transcription factor activation, providing HSCs with critical surviving cues. In agreement, recue of Ret null progenitors was efficiently achieved in vivo by forcing the expression of RET downstream targets, Bcl2 or Bcl2l1. Thus, RET activation improves HSC survival and in vivo transplantation efficiency, unveiling exciting new possibilities in transplantation and HSC ex vivo expansion. In addition, our work demonstrates that HSC use neurotrophic factors to regulate and maintain their fitness. RET signalling molecules are also expressed in thymocytes, more specifically, we found their expression in CD4/CD8 double negative thymocytes (DN). Nevertheless, ablation of Ret or it co-receptors Gfra1 and Gfra2 had a minor impact in foetal thymopoiesis. In agreement, Ret conditional knockout mice had similar thymocyte development and fitness when compared to their WT counterparts. Thus, while RET signalling is critical to HSC function, it is dispensable for T cell development in vivo. Altogether, our work show that molecular mechanisms usually assigned to specific tissues, can be more widely used by unrelated cell types, such as haematopoietic stem cells. Our findings also illustrate how a same signalling pathway can be regulated to originate different cell responses. Unlike several neuronal populations that use GFLs depending on the specific expressed co-receptor, HSCs express multiple RET coreceptors and respond to GFLs in a redundant fashion. There is increasing evidence that nervous signals can control haematopoiesis, namely by regulating osteoblast and mesenchymal stem cell function in HSC niches. Herein, we show that neurons and HSC employ common regulatory mechanisms. Thus, our work paves the way to further studies employing neurotrophic factors in HSC expansion and transplantation protocols.

Objective. CD8+ T cells are part of the T cell pool infiltrating the synovium in rheumatoid arthritis (RA). However, their role in the pathogenesis of RA has not been fully delineated. Using the K/BxN mouse model of spontaneous chronic arthritis, which shares many similarities with RA, we studied the potential of CD8 T cell depletion with monoclonal antibodies (mAb) to stop and reverse the progression of experimental arthritis. Methods. CD8+ T cells from the blood and articular infiltrate of K/BxN mice were characterized for cell surface phenotypic markers and for cytokine production. Additionally, mice were treated with specific anti-CD8 mAb (YTS105 and YTS169.4), with and without thymectomy. Results. CD8+ T cells from the peripheral blood and joints of K/BxN mice were mainly CD69+ and CD62L-CD27+ T cells expressing proinflammatory cytokines (interferon-γ [IFNγ], tumor necrosis factor α [TNFα], interleukin-17a [IL-17A], and IL-4), and granzyme B. In mice receiving anti-CD8 mAb, the arthritis score improved 5 days after treatment. Recovery of the CD8+ T cells was associated with a new increase in the arthritis score after 20 days. In thymectomized and anti-CD8 Ab–treated mice, the arthritis score improved permanently. Histologic analysis showed an absence of inflammatory infiltrate in the anti-CD8 mAb–treated mice. In anti-CD8 mAb–treated mice, the serologic levels of TNFα, IFNγ, IL-6, and IL-5 normalized. The levels of the disease-related anti–glucose-6-phosphate isomerase antibodies did not change. Conclusion. These results indicate that synovial activated effector CD8 T cells locally synthesize proinflammatory cytokines (IFNγ, TNFα, IL-17, IL-6) and granzyme B in the arthritic joint, thus playing a pivotal role in maintaining chronic synovitis in the K/BxN mouse model of arthritis.

Tumour immunosurveillance postulates that immune cells are able to eliminate transformed cells, as much as they eliminate pathogens or pathogeninfected cells. This theory constitutes the basis of cancer immunotherapy which explores anti-tumour properties of the immune system (both innate and adaptive) for the treatment of human malignancies. γδ T cells are innate-like lymphocytes that account for a small percentage (1- 10%) of human peripheral blood lymphocytes, but represent the majority of T cells in epithelial tissues. Several properties make γδ T cells attractive targets for cancer immunotherapy, namely their MHC-unrestricted cytotoxicity and unique responsiveness to clinical grade available (phospho) agonists. Despite the promise of γδ T cells in cancer immunotherapy, the clinical success has been limited by low percentages of objective responses. This urges more research on the mechanisms that govern the interactions between γδ T cells and tumours. One of the major gaps in the γδ T cell field is the lack of mechanistic knowledge on tumour cell recognition. We decided to analyze which molecules determine haematological tumour recognition by Vγ9Vδ2 T cells, the major γδ T cell subsets in human peripheral blood. We observed widely variable susceptibility of leukaemia and lymphoma cell lines to Vγ9Vδ2 T cell-mediated cytotoxicity. For those tumours that were efficiently targeted by Vγ9Vδ2 T cells, we found that this required the NK receptor NKG2D, but not the signature T cell receptor (TCR). We then analyzed the expression of NKG2D ligands in susceptible or resistant tumour cells lines, and observed that ULBP1 expression (both at the mRNA level and protein level) segregated with susceptible targets. Through a series of loss- and gain-offunction assays, we demonstrated that ULBP1 constitutes a non-redundant determinant of leukaemia and lymphoma cell recognition by human γδ T cells. Importantly, we observed a dramatic heterogeneity of ULBP1 expression in primary samples obtained from leukaemia and lymphoma patients, which can thus contribute to the highly variable outcomes of γδ T cell-based clinical trials in haematological tumours. Another important limitation to the modulation of γδ T cells in cancer immunotherapy is the lack of molecular cues that direct γδ T cell migration to tumours. Given the pivotal role played by chemokines and their receptors in leukocyte migration, we investigated which chemokines could determine γδ T cell recruitment to tumours. We used the “gold standard” pre-clinical transplantable B16 melanoma model and compared chemokine composition of tumour extracts from WT and TCRδ-deficient mice, and observed an accumulation of the CCR2 ligands, CCL2 and CCL12, in tumour extracts from TCRδ-deficient animals. Interestingly, the comparison of WT and CCR2-deficient hosts revealed increased tumour growth in CCR2-/- mice. Critically, we showed that tumours from CCR2-/- (as well as CCL2-/-) mice contained significantly less infiltrating γδ T cells compared to WT tumours, whereas other T cell populations, particularly CD4+ and CD8+ T cells, were not affected. We also analysed myeloid populations, namely macrophages, neutrophils and myeloid-derived suppressor cells (MDSCs), and observed that, as expected from previous literature, these leukocyte populations were reduced in tumours from CCR2-/- mice. Considering that myeloid cell infiltration into tumours is usually associated with poor prognosis, it is noticeable that CCR2-/- mice displayed increased tumour growth when compared to WT mice. Furthermore, as TCRδ-deficient mice also showed increased tumour burden (compared to WT mice), our data suggests a novel protective role for the CCR2/ CCL2 chemokine pathway through the recruitment of cytotoxic γδ T cells. Considering the significant differences between murine and human γδ T cells, and trying to translate these findings into the human setting, we also investigated if human γδ T cells relied on CCR2 for migration. We observed that the Vδ1 subset expresses CCR2 and migrates towards CCL2 in vitro; by contrast with the Vδ2 population that lacks CCR2 expression, even after activation. Moreover, we observed a dramatic variety of CCL2 levels in several cancer types, with some overexpressing and other downmodulating CCL2 when compared to healthy tissue controls. These data collectively highlight the importance of correlating Vδ1 T cell infiltration with CCL2 expression in situ in future cancer clinical studies. While the potent anti-tumour properties of γδ T cells have been widely demonstrated, some recent reports have implied a pro-tumour role for γδ T cells. We therefore set out to investigate which conditions determine the anti- versus protumour properties of γδ T cells. By comparing two different murine tumour models, B16 melanoma and ID8 ovarian carcinoma, we proposed that the pro-tumour role of γδ T cells may be determined by IL-17 production. TCRδ-deficient mice showed reduced survival (compared to WT) when challenged with B16 tumours, but enhanced survival when challenged with ID8 tumours. We demonstrated an accumulation of IL-17-producing γδ T cells in the peritoneal cavity of ID8 tumour bearing mice that seemed to dominate over anti-tumour IFN-γ production. Importantly, the major source of IL-17 in the ID8 tumour model were γδ T cells, and these expressed higher levels of IL-17 (on a per cell basis) than their CD4+T cell counterparts. In summary, in this thesis we have characterized some key features of γδ T cells in tumour immunosurveillance, namely leukaemia/ lymphoma cell recognition; migration and tumour infiltration; and putative pro-tumour properties. We believe these findings make an important contribution to our understanding of γδ T cell function, and may help to improve current cancer immunotherapy protocols based on γδ T cell activation.

γδ T cells are regarded as promising effector lymphocytes for next-generation cancer immunotherapies. In spite of being relatively rare in human peripheral blood, γδ T cells are more abundant in epithelial tissues where many tumors develop, and have been shown to actively participate in anticancer immunity as cytotoxic cells or as "type 1" immune orchestrators. A major asset of γδ T cells for tackling advanced cancers is their independence from antigen presentation via the major histocompatibility complex, which clearly sets them apart from conventional αβ T cells. Here we discuss the main therapeutic strategies based on human γδ T cells. These include antibody-based bispecific engagers and adoptive cell therapies, either focused on the Vδ1+ or Vδ2+ γδ T-cell subsets, which can be expanded selectively and differentiated or engineered to maximize their antitumor functions. We review the preclinical data that supports each of the therapeutic strategies under development; and summarize the clinical trials being pursued towards establishing γδ T cell-based treatments for solid and hematological malignancies.

A classic T-cell phenotype in systemic lupus erythematosus (SLE) is the downregulation and replacement of the CD3ζ chain that alters T-cell receptor signaling. However, genetic associations with SLE in the human CD247 locus that encodes CD3ζ are not well established and require replication in independent cohorts. Our aim was therefore to examine, localize and validate CD247-SLE association in a large multiethnic population. We typed 44 contiguous CD247 single-nucleotide polymorphisms (SNPs) in 8922 SLE patients and 8077 controls from four ethnically distinct populations. The strongest associations were found in the Asian population (11 SNPs in intron 1, 4.99 × 10(-4) < P < 4.15 × 10(-2)), where we further identified a five-marker haplotype (rs12141731-rs2949655-rs16859085-rs12144621-rs858554; G-G-A-G-A; P(hap) = 2.12 × 10(-5)) that exceeded the most associated single SNP rs858554 (minor allele frequency in controls = 13%; P = 4.99 × 10(-4), odds ratio = 1.32) in significance. Imputation and subsequent association analysis showed evidence of association (P < 0.05) at 27 additional SNPs within intron 1. Cross-ethnic meta-analysis, assuming an additive genetic model adjusted for population proportions, showed five SNPs with significant P-values (1.40 × 10(-3) < P< 3.97 × 10(-2)), with one (rs704848) remaining significant after Bonferroni correction (P(meta) = 2.66 × 10(-2)). Our study independently confirms and extends the association of SLE with CD247, which is shared by various autoimmune disorders and supports a common T-cell-mediated mechanism.

In human systemic lupus erythematosus (SLE), diverse autoantibodies accumulate over years before disease manifestation. Unaffected relatives of SLE patients frequently share a sustained production of autoantibodies with indiscriminable specificity, usually without ever acquiring the disease. We studied relations of IgG autoantibody profiles and peripheral blood activated regulatory T-cells (aTregs), represented by CD4(+)CD25(bright) T-cells that were regularly 70-90% Foxp3(+). We found consistent positive correlations of broad-range as well as specific SLE-associated IgG with aTreg frequencies within unaffected relatives, but not patients or unrelated controls. Our interpretation: unaffected relatives with shared genetic factors compensated pathogenic effects by aTregs engaged in parallel with the individual autoantibody production. To study this further, we applied a novel analytic approach named coreferentiality that tests the indirect relatedness of parameters in respect to multivariate phenotype data. Results show that independently of their direct correlation, aTreg frequencies and specific SLE-associated IgG were likely functionally related in unaffected relatives: they significantly parallelled each other in their relations to broad-range immunoblot autoantibody profiles. In unaffected relatives, we also found coreferential effects of genetic variation in the loci encoding IL-2 and CD25. A model of CD25 functional genetic effects constructed by coreferentiality maximization suggests that IL-2-CD25 interaction, likely stimulating aTregs in unaffected relatives, had an opposed effect in SLE patients, presumably triggering primarily T-effector cells in this group. Coreferentiality modeling as we do it here could also be useful in other contexts, particularly to explore combined functional genetic effects.

Virus host evasion genes are ready-made tools for gene manipulation and therapy. In this work we have assessed the impact in vivo of the evasion gene A238L of the African Swine Fever Virus, a gene which inhibits transcription mediated by both NF-κB and NFAT. The A238L gene has been selectively expressed in mouse T lymphocytes using tissue specific promoter, enhancer and locus control region sequences for CD2. The resulting two independently derived transgenic mice expressed the transgene and developed a metastasic, angiogenic and transplantable CD4(+)CD8(+)CD69(-) lymphoma. The CD4(+)CD8(+)CD69(-) cells also grew vigorously in vitro. The absence of CD69 from the tumour cells suggests that they were derived from T cells at a stage prior to positive selection. In contrast, transgenic mice similarly expressing a mutant A238L, solely inhibiting transcription mediated by NF-κB, were indistinguishable from wild type mice. Expression of Rag1, Rag2, TCRβ-V8.2, CD25, FoxP3, Bcl3, Bcl2 l14, Myc, IL-2, NFAT1 and Itk, by purified CD4(+)CD8(+)CD69(-) thymocytes from A238L transgenic mice was consistent with the phenotype. Similarly evaluated expression profiles of CD4(+)CD8(+) CD69(-) thymocytes from the mutant A238L transgenic mice were comparable to those of wild type mice. These features, together with the demonstration of (mono-)oligoclonality, suggest a transgene-NFAT-dependent transformation yielding a lymphoma with a phenotype reminiscent of some acute lymphoblastic lymphomas.

CKD is associated with a complex state of immune dysfunction characterized by immune depression, predisposing patients to infections, and immune activation, resulting in inflammation that associates with higher risk of cardiovascular disease. Physical exercise may enhance immune function and exert anti-inflammatory effects, but such effects are unclear in CKD. We investigated the separate effects of acute and regular moderate-intensity aerobic exercise on neutrophil degranulation (elastase release), activation of T lymphocytes (CD69 expression) and monocytes (CD86 and HLA-DR expression), and plasma inflammatory markers (IL-6, IL-10, soluble TNF-receptors, and C-reactive protein) in patients with predialysis CKD. A single 30-minute (acute) bout of walking induced a normal pattern of leukocyte mobilization and had no effect on T-lymphocyte and monocyte activation but improved neutrophil responsiveness to a bacterial challenge in the postexercise period. Furthermore, acute exercise induced a systemic anti-inflammatory environment, evidenced by a marked increase in plasma IL-10 levels (peaked at 1 hour postexercise), that was most likely mediated by increased plasma IL-6 levels (peaked immediately postexercise). Six months of regular walking exercise (30 min/d for 5 times/wk) exerted anti-inflammatory effects (reduction in the ratio of plasma IL-6 to IL-10 levels) and a downregulation of T-lymphocyte and monocyte activation, but it had no effect on circulating immune cell numbers or neutrophil degranulation responses. Renal function, proteinuria, and BP were also unaffected. These findings provide compelling evidence that walking exercise is safe with regard to immune and inflammatory responses and has the potential to be an effective anti-inflammatory therapy in predialysis CKD.