Systemic inflammatory Th1 cytokines during Trypanosoma cruzi infection disrupt the typical anatomical cell distribution and phenotypic/functional characteristics of various cell subsets within the thymus.

The thymus plays a crucial role in T cell differentiation, a complex process influenced by various factors such as antigens, the microenvironment and thymic architecture. The way the thymus resolves infections is critical, as chronic persistence of microbes or inflammatory mediators can obstruct the differentiation. Here, we illustrate that following inflammatory T helper 1 infectious processes like those caused by Candida albicans or Trypanosoma cruzi, single positive thymocytes adopt a mature phenotype. Further investigations focused on T. cruzi infection, reveal a substantial existence of CD44+ cells in both the cortical and medullary areas of the thymus at the onset of infection. This disturbance coincides with heightened interferon gamma (IFNγ) production by thymocytes and an increased cytotoxic capacity against T. cruzi-infected macrophages. Additionally, we observe a reduced exportation capacity in T. cruzi-infected mice. Some alterations can be reversed in IFNγ knockout mice (KO). Notably, the majority of these effects can be replicated by systemic expression of interleukin (IL)-12+IL-18, underlining the predominantly inflammatory rather than pathogen-specific nature of these phenomena. Understanding the mechanisms through which systemic inflammation disrupts normal T cell development, as well as subsequent T cell exportation to secondary lymphoid organs (SLO) is pivotal for comprehending susceptibility to diseases in different pathological scenarios.

COVID-19 patients display changes in lymphocyte subsets with a higher frequency of dysfunctional CD8lo T cells associated with disease severity.

This work examines cellular immunity against SARS-CoV-2 in patients from Córdoba, Argentina, during two major waves characterized by different circulating viral variants and different social behavior. Using flow cytometry, we evaluated the main lymphocyte populations of peripheral blood from hospitalized patients with moderate and severe COVID-19 disease. Our results show disturbances in the cellular immune compartment, as previously reported in different cohorts worldwide. We observed an increased frequency of B cells and a significant decrease in the frequency of CD3+ T cells in COVID-19 patients compared to healthy donors (HD). We also found a reduction in Tregs, which was more pronounced in severe patients. During the first wave, the frequency of GZMB, CD107a, CD39, and PD-1-expressing conventional CD4+ T (T conv) cells was significantly higher in moderate and severe patients than in HD. During the second wave, only the GZMB+ T conv cells of moderate and severe patients increased significantly. In addition, these patients showed a decreased frequency in IL-2-producing T conv cells. Interestingly, we identified two subsets of circulating CD8+ T cells with low and high CD8 surface expression in both HD and COVID-19 patients. While the percentages of CD8hi and CD8lo T cells within the CD8+ population in HD are similar, a significant increase was observed in CD8lo T cell frequency in COVID-19 patients. CD8lo T cell populations from HD as well as from SARS-CoV-2 infected patients exhibited lower frequencies of the effector cytokine-producing cells, TNF, IL-2, and IFN-γ, than CD8hi T cells. Interestingly, the frequency of CD8lo T cells increased with disease severity, suggesting that this parameter could be a potential marker for disease progression. Indeed, the CD8hi/CD8lo index helped to significantly improve the patient's clinical stratification and disease outcome prediction. Our data support the addition of, at least, a CD8hi/CD8lo index into the panel of biomarkers commonly used in clinical labs, since its determination may be a useful tool with impact on the therapeutic management of the patients.

Different cytokine and chemokine profiles in hospitalized patients with COVID-19 during the first and second outbreaks from Argentina show no association with clinical comorbidities.

Background: COVID-19 severity has been linked to an increased production of inflammatory mediators called "cytokine storm". Available data is mainly restricted to the first international outbreak and reports highly variable results. This study compares demographic and clinical features of patients with COVID-19 from Córdoba, Argentina, during the first two waves of the pandemic and analyzes association between comorbidities and disease outcome with the "cytokine storm", offering added value to the field. Methods: We investigated serum concentration of thirteen soluble mediators, including cytokines and chemokines, in hospitalized patients with moderate and severe COVID-19, without previous rheumatic and autoimmune diseases, from the central region of Argentina during the first and second infection waves. Samples from healthy controls were also assayed. Clinical and biochemical parameters were collected. Results: Comparison between the two first COVID-19 waves in Argentina highlighted that patients recruited during the second wave were younger and showed less concurrent comorbidities than those from the first outbreak. We also recognized particularities in the signatures of systemic cytokines and chemokines in patients from both infection waves. We determined that concurrent pre-existing comorbidities did not have contribution to serum concentration of systemic cytokines and chemokines in COVID-19 patients. We also identified immunological and biochemical parameters associated to inflammation which can be used as prognostic markers. Thus, IL-6 concentration, C reactive protein level and platelet count allowed to discriminate between death and discharge in patients hospitalized with severe COVID-19 only during the first but not the second wave. Conclusions: Our data provide information that deepens our understanding of COVID-19 pathogenesis linking demographic features of a COVID-19 cohort with cytokines and chemokines systemic concentration, presence of comorbidities and different disease outcomes. Altogether, our findings provide information not only at local level by delineating inflammatory/anti-inflammatory response of patients but also at international level addressing the impact of comorbidities and the infection wave in the variability of cytokine and chemokine production upon SARS-CoV-2 infection.

Exploring the immunomodulatory role of virtual memory CD8+ T cells: Role of IFN gamma in tumor growth control.

Virtual memory CD8+ T cells (TVM) have been described as cells with a memory-like phenotype but without previous antigen (Ag) exposure. TVM cells have the ability to respond better to innate stimuli rather than by TCR engagement, producing large amounts of interferon gamma (IFNγ) after stimulation with interleukin (IL)-12 plus IL-18. As a result of the phenotypic similarity, TVM cells have been erroneously included in the central memory T cell subset for many years. However, they can now be discriminated via the CD49d receptor, which is up-regulated only on conventional memory T cells (TMEM) and effector T cells (TEFF) after specific cognate Ag recognition by a TCR. In this work we show that systemic expression of IL-12 plus IL-18 induced an alteration in the normal TVM vs TMEM/TEFF distribution in secondary lymphoid organs and a preferential enrichment of TVM cells in the melanoma (B16) and the pancreatic ductal adenocarcinoma (KPC) tumor models. Using our KPC bearing OT-I mouse model, we observed a significant increase in CD8+ T cell infiltrating the tumor islets after IL-12+IL-18 stimulation with a lower average speed when compared to those from control mice. This finding indicates a stronger interaction of T cells with tumor cells after cytokine stimulation. These results correlate with a significant reduction in tumor size in both tumor models in IL-12+IL-18-treated OT-I mice compared to control OT-I mice. Interestingly, the absence of IFNγ completely abolished the high antitumor capacity induced by IL-12+IL-18 expression, indicating an important role for these cytokines in early tumor growth control. Thus, our studies provide significant new information that indicates an important role of TVM cells in the immune response against cancer.

TIM3 Expression in Anaplastic-Thyroid-Cancer-Infiltrating Macrophages: An Emerging Immunotherapeutic Target.

Anaplastic thyroid cancer (ATC) is a clinically aggressive form of undifferentiated thyroid cancer with limited treatment options. Immunotherapy for patients with ATC remains challenging. Tumor-associated macrophages (TAMs) constitute over 50% of ATC-infiltrating cells, and their presence is associated with a poor prognosis. Consequently, the development of new therapies targeting immune checkpoints in TAMs is considered a promising therapeutic approach for ATC. We have previously shown that soluble factors secreted by ATC cells induced pro-tumor M2-like polarization of human monocytes by upregulating the levels of the inhibitory receptor TIM3. Here, we extended our observations on ATC-cell-induced xenograft tumors. We observed a large number of immune cells infiltrating the ATC xenograft tumors. Significantly, 24-28% of CD45+ immune cells were macrophages (CD11b+ F4/80+). We further showed that 40% of macrophages were polarized toward a M2-like phenotype, as assessed by CD206 expression and by a significant increase in the Arg1/iNOS (M2/M1) ratio. Additionally, we found that ATC xenograft tumors had levels of TIM3 expression when determined by RT-PCR and immunofluorescence assays. Interestingly, we detected the expression of TIM3 in macrophages in ATC tumors by flow cytometry assays. Furthermore, TIM3 expression correlated with macrophage marker expression in human ATC. Our studies show that TIM3 is a newly identified immune checkpoint in macrophages. Since TIM3 is known as a negative immune regulator, it should be considered as a promising immunotherapeutic target for ATC.

Virtual Memory CD8+ T Cells: Origin and Beyond.

The presence of CD8+ T cells with a memory phenotype in nonimmunized mice has been noted for decades, but it was not until about 2 decades ago that they began to be studied in greater depth. Currently called virtual memory CD8+ T cells, they consist of a heterogeneous group of cells with memory characteristics, without any previous contact with their specific antigens. These cells were identified in mice, but a few years ago, a cell type with characteristics equivalent to the murine ones was described in healthy humans. In this review, we address the different aspects of its biology mainly developed in murine models and what is currently known about its cellular equivalent in humans.

The Thymus in Chagas Disease: Molecular Interactions Involved in Abnormal T-Cell Migration and Differentiation.

Chagas disease, caused by the protozoan parasite T. cruzi, is a prevalent parasitic disease in Latin America. Presently, it is spreading around the world by human migration, thus representing a new global health issue. Chronically infected individuals reveal a dissimilar disease progression: while nearly 60% remain without apparent disease for life, 30% develop life-threatening pathologies, such as chronic chagasic cardiomyopathy (CCC) or megaviscerae. Inflammation driven by parasite persistence seems to be involved in the pathophysiology of the disease. However, there is also evidence of the occurrence of autoimmune events, mainly caused by molecular mimicry and bystander activation. In experimental models of disease, is well-established that T. cruzi infects the thymus and causes locally profound structural and functional alterations. The hallmark is a massive loss of CD4+CD8+ double positive (DP) thymocytes, mainly triggered by increased levels of glucocorticoids, although other mechanisms seem to act simultaneously. Thymic epithelial cells (TEC) exhibited an increase in extracellular matrix deposition, which are related to thymocyte migratory alterations. Moreover, medullary TEC showed a decreased expression of AIRE and altered expression of microRNAs, which might be linked to a disrupted negative selection of the T-cell repertoire. Also, almost all stages of thymocyte development are altered, including an abnormal output of CD4-CD8- double negative (DN) and DP immature and mature cells, many of them carrying prohibited TCR-Vß segments. Evidence has shown that DN and DP cells with an activated phenotype can be tracked in the blood of humans with chronic Chagas disease and also in the secondary lymphoid organs and heart of infected mice, raising new questions about the relevance of these populations in the pathogenesis of Chagas disease and their possible link with thymic alterations and an immunoendocrine imbalance. Here, we discuss diverse molecular mechanisms underlying thymic abnormalities occurring during T. cruzi infection and their link with CCC, which may contribute to the design of innovative strategies to control Chagas disease pathology.

Thymic expression of IL-4 and IL-15 after systemic inflammatory or infectious Th1 disease processes induce the acquisition of "innate" characteristics during CD8+ T cell development.

Innate CD8+ T cells express a memory-like phenotype and demonstrate a strong cytotoxic capacity that is critical during the early phase of the host response to certain bacterial and viral infections. These cells arise in the thymus and depend on IL-4 and IL-15 for their development. Even though innate CD8+ T cells exist in the thymus of WT mice in low numbers, they are highly enriched in KO mice that lack certain kinases, leading to an increase in IL-4 production by thymic NKT cells. Our work describes that in C57BL/6 WT mice undergoing a Th1 biased infectious disease, the thymus experiences an enrichment of single positive CD8 (SP8) thymocytes that share all the established phenotypical and functional characteristics of innate CD8+ T cells. Moreover, through in vivo experiments, we demonstrate a significant increase in survival and a lower parasitemia in mice adoptively transferred with SP8 thymocytes from OT I-T. cruzi-infected mice, demonstrating that innate CD8+ thymocytes are able to protect against a lethal T. cruzi infection in an Ag-independent manner. Interestingly, we obtained similar results when using thymocytes from systemic IL-12 + IL-18-treated mice. This data indicates that cytokines triggered during the acute stage of a Th1 infectious process induce thymic production of IL-4 along with IL-15 expression resulting in an adequate niche for development of innate CD8+ T cells as early as the double positive (DP) stage. Our data demonstrate that the thymus can sense systemic inflammatory situations and alter its conventional CD8 developmental pathway when a rapid innate immune response is required to control different types of pathogens.

Type I IFNs Are Required to Promote Central Nervous System Immune Surveillance through the Recruitment of Inflammatory Monocytes upon Systemic Inflammation.

Brain-resident microglia and peripheral migratory leukocytes play essential roles in shaping the immune response in the central nervous system. These cells activate and migrate in response to chemokines produced during active immune responses and may contribute to the progression of neuroinflammation. Herein, we addressed the participation of type I-II interferons in the response displayed by microglia and inflammatory monocytes to comprehend the contribution of these cytokines in the establishment and development of a neuroinflammatory process. Following systemic lipopolysaccharide (LPS) challenge, we found glial reactivity and an active recruitment of CD45hi leukocytes close to CD31+ vascular endothelial cells in circumventricular organs. Isolated CD11b+ CD45hi Ly6Chi Ly6G--primed inflammatory monocytes were able to induce T cell proliferation, unlike CD11b+ CD45lo microglia. Moreover, ex vivo re-stimulation with LPS exhibited an enhancement of T cell proliferative response promoted by inflammatory monocytes. These myeloid cells also proved to be recruited in a type I interferon-dependent fashion as opposed to neutrophils, unveiling a role of these cytokines in their trafficking. Together, our results compares the phenotypic and functional features between tissue-resident vs peripheral recruited cells in an inflamed microenvironment, identifying inflammatory monocytes as key sentinels in a LPS-induced murine model of neuroinflammation.

Intestinal mononuclear cells primed by systemic interleukin-12 display long-term ability to aggravate colitis in mice.

To address whether the burst of systemic interleukin-12 (IL-12) influences intestinal inflammation elicited by luminal stimuli, we induced IL-12 release by cDNA injection in C57BL/6 mice and simultaneously started dextran sulphate sodium administration. The sequence of the inflammatory response triggered by IL-12 release was characterized by assessing myeloperoxidase activity and histological damage in colon samples on days 1, 3, 5 and 7 after colitis induction. To evaluate the persistence of IL-12 priming, colitis was induced in mice 7 or 60 days after cDNA injection. Under IL-12 influence, the development of acute colitis presented a faster and selective infiltration of inflammatory mononuclear cells in the lamina propria. Recruitment was driven by systemic cytokines rather than luminal antigens. Interestingly, when colitis was triggered 7 or 60 days after the cytokine storm, cells maintained the ability to worsen clinical signs of intestinal inflammation. Together, a systemic IL-12 burst effectively primed intestinal cells that became more prone to develop inflammatory responses. Activation was long-lasting because intestinal cells maintained their inflammatory potential and their ability to aggravate colitis.

Systemic IL-12 burst expands intestinal T-lymphocyte subsets bearing the α4 ß7 integrin in mice.

The intestinal immune system is complex and displays unique anatomic and functional characteristics. Numerous immune cell subsets are located beneath the epithelial barrier and their activity is highly regulated. Using hydrodynamic shear of IL-12 cDNA to achieve systemic expression of IL-12 in mice, we evaluated the effect of a transient burst of this cytokine on the activation status of T cells from Peyer's patches (PPs), mesenteric lymph nodes (MLNs), and colonic lamina propria (LP). Following systemic IL-12 release, intestinal T lymphocytes became activated, exhibiting a CD44(high) CD62L(-) phenotype. After 5 days of the cytokine burst, the frequency of α4ß7(+) CD4(+) and CD8(+) cells increased, and CD8(+) α4ß7(+) cells mainly expressed T bet, a critical regulator of the Th1 differentiation program. The incremental increase in α4ß7 expression involved the IL-12 receptor-signal transducer and activator of transcription (STAT)-4 axis, and occurred independently of IFN-γ, IL-4, IL-10, and TNF-α signaling. Moreover, IL-12 priming exacerbated the outcome of acute dextran sodium sulphate (DSS)-induced colitis with higher scores of weight loss, blood in stool, and diarrhea and lower hematocrit. Together, our findings demonstrate that systemic polarizing signals could effectively expand the number of effector cells able to home to the LP and contribute to local inflammation.

Candida albicans up-regulates the Fas-L expression in liver Natural Killer and Natural Killer T cells.

After Candida albicans arrival to the liver, the local production of proinflammatory cytokines and the expanded intrahepatic lymphocytes (IHL) can be either beneficial or detrimental to the host. Herein we explored the balance between protective inflammatory reaction and liver damage, focusing our study on the contribution of TNF-α and Fas-Fas-L pathways in the hepatocellular apoptosis associated to C. albicans infection. A robust tissue reaction and a progressive increase of IL-1ß, IL-6 and TNF-α were observed in infected animals. Blocking the biological activity of TNF-α did not modify the number of apoptotic cells observed in C. albicans infected animals. Fas-L molecule was up regulated on purified hepatic mononuclear cells and its expression progressed with the infection. In the IHL compartment, the absolute number of Fas-L+ NK and NKT cells increased on days 1 and 3 of the infection. C. albicans was also able to up regulate Fas-L expression in normal liver NK and NKT cells after in vitro contact. The innate receptor TLR2 was involved in this phenomenon. In the interplay between host factors and evasion strategies exploited by pathogens, the mechanism supported here could represent an additional way that allows this fungus to circumvent protective immune responses in the liver.

Abrogation of TNFα production during cancer immunotherapy is crucial for suppressing side effects due to the systemic expression of IL-12.

For more than a decade, the cytokine interleukin-12 (IL-12) has been utilized, either alone or in combination with other drugs, as a treatment for cancer. The numerous anti-tumor properties of IL-12 still generate interest in the clinical use of this cytokine, even though it has demonstrated toxicity when administrated systemically. As an approach to overcome this toxicity, numerous laboratories have attempted to induce IL-12 expression at the site of the tumor. However for tumors that are difficult to remove surgically or for the treatment of disseminated metastases, systemic expression of this cytokine still remains as the most efficient method of administration. Nevertheless, finding alternative approaches for the use of IL-12 in the treatment of cancer and unraveling the basis of IL-12-side effects remain a challenge. In the present work we demonstrate that systemic expression of IL-12 through hydrodynamic injection of IL-12 cDNA is able to induce different types of liver lesions associated with a toxic pathology. However we report here that hepatic toxicity is diminished and survival of mice enhanced in the absence of tumor necrosis factor alpha (TNFα). This observation is in contrast to several murine models and clinical trials that postulate interferon gamma (IFNγ) as the main cytokine responsible for IL-12 toxicity. Moreover, our work demonstrates that when IL-12 cDNA is co-injected with IL-18 cDNA or when mice are pre-treated with a low dose of IL-12 cDNA prior to receiving a high dose of IL-12 cDNA, systemic levels of TNFα are almost completely abrogated, resulting in improved survival and less hepatic damage. Importantly, abrogation of TNFα signaling does not affect the strong anti-tumor activity of IL-12. Thus, neutralizing TNFα with antagonists already approved for human use offers a promising approach to abrogate IL-12 side effects during the use of this cytokine for the treatment of cancer.

MCP-1/CCR2 interactions direct migration of peripheral B and T lymphocytes to the thymus during acute infectious/inflammatory processes.

Mature lymphocyte immigration into the thymus has been documented in mouse, rat, and pig models, and highly increases when cells acquire an activated phenotype. Entrance of peripheral B and T cells into the thymus has been described in healthy and pathological situations. However, it has not been proposed that leukocyte recirculation to the thymus could be a common feature occurring during the early phase of a Th1 inflammatory/infectious process when a large number of peripheral cells acquire an activated phenotype and the cellularity of the thymus is seriously compromised. The data we present here demonstrate that in well-established Th1 models triggered by different types of immunogens, for example, LPS treatment (a bacterial product), Candida albicans infection (a fungus), and after Trypanosoma cruzi infection (a parasite), a large number of mature peripheral B and T cells enter the thymus. This effect is dependent on, but not exclusive of, the available space in the thymus. Our data also demonstrate that MCP-1/CCR2 (where MCP-1 is monocyte chemoattractant protein-1) interaction is responsible for the infiltration of peripheral cells to the thymus in these Th1-inflammatory/infectious situations. Finally, systemic expression of IL-12 and IL-18 produced during the inflammatory process is ultimately responsible for these migratory events.

Abrogation of spontaneous liver tolerance during immune response to Candida albicans: contribution of NKT and hepatic mononuclear cells.

Hepatic mononuclear cells (HMC) are a heterogeneous population with innate immune properties involved in the response to several pathogens. Herein, during the primary infection with Candida albicans, we observed dynamic changes in CD3+, NK+ and NKT+ intrahepatic lymphoid subsets and a significant increase in the absolute number of antigen-presenting cells (APC). The liver tolerogenic microenvironment sustained by higher levels of IL-10, transforming growth factor-ß and IL-4 was severely modified upon the robust IFN-γ production after the fungal colonization. NKT cells purified from infected animals released significant amounts of IFN-γ and the production of this cytokine was exacerbated after a second contact with the fungus. Interestingly, C. albicans per se was unable to activate tolerogenic NKT cells from naive animals. In vitro experiments performed with HMC cells depleted of the CD11b/c+ population revealed that in the absence of APC, NKT cells are unable to produce IFN-γ in response to C. albicans. Our findings constitute the first evidence that this innate lymphocyte population is involved in the pathogenesis of C. albicans infection.

Immune neuroendocrine interactions during a fungal infection in immunocompetent or immunosuppressed hosts.

The yeast Candida albicans belongs to the microflora of healthy individuals, although it can infect a variety of tissues ensuing changes in the host's immune status. To evaluate the effect of neuroendocrine input on the early immune response during the fungal infection, we use a 3-day paradigm of chronic varied stress in Wistar rats infected with C. albicans. We find that stress mediators contribute to the spread of the fungus and downregulate critical functions of phagocytic cells at the infection site. Phenotypic and functional alterations of effector cells account for the decreased resistance to candidiasis and condition the development of the adaptive response. Stressed hosts exhibit a higher fungal burden in kidneys and livers associated with hyphal forms. The hepatic inflammatory reaction is compromised with severe steatosis, increment of functional enzymes, marked lipid peroxidation and hepatocyte apoptosis. Moreover, infection-related sickness symptoms are significantly increased by exposure to stress with anorexia, weight loss, lack of leptin and depletion of glycogen depots. Food deprivation exacerbates the liver injury. Stress mediators perturb the complex immune and metabolic program that operates early during fungal spread and promotes severe tissue damage.

Coexpression of IL-18 strongly attenuates IL-12-induced systemic toxicity through a rapid induction of IL-10 without affecting its antitumor capacity.

IL-12 is an excellent candidate for the treatment of cancer due to its ability to drive strong antitumor responses. Recombinant IL-12 protein is currently used in cancer patients; however, systemic expression of rIL-12 presents disadvantages including cost and dose limitation due to its toxicity. In this study, we used hydrodynamic shear of cDNA as a tool to achieve systemic expression of IL-12. We found that sustained but toxic levels of serum IL-12 could be generated in 6- to 7-wk-old B6 mice after a single injection of the cDNA. Unexpectedly, we observed that when IL-12 cDNA is coinjected with IL-18 cDNA, IL-12 antitumor activity was maintained, but there was a significant attenuation of IL-12 toxicity, as evidenced by a greater survival index and a diminution of liver enzymes (ALT and AST). Interestingly, after IL-12 plus IL-18 cDNA administration, more rapid and higher IL-10 levels were observed than after IL-12 cDNA treatment alone. To understand the mechanism of protection, we coinjected IL-12 plus IL-10 cDNAs and observed an increase in survival that correlated with diminished serum levels of the inflammatory cytokines TNF-alpha and IFN-gamma. Confirming the protective role of early IL-10 expression, we observed a significant decrease in survival in IL-10 knockout mice or IL-10R-blocked B6 mice after IL-12 plus IL-18 treatment. Thus, our data demonstrate that the high and early IL-10 expression induced after IL-12 plus IL-18 cDNA treatment is critical to rapidly attenuate IL-12 toxicity without affecting its antitumor capacity. These data could highly contribute to the design of more efficient/less toxic protocols for the treatment of cancer.

Hematopoiesis and thymic apoptosis are not affected by the loss of Cdk2.

Cell cycle regulation is essential for proper homeostasis of hematopoietic cells. Cdk2 is a major regulator of S phase entry, is activated by mitogenic cytokines, and has been suggested to be involved in antigen-induced apoptosis of T lymphocytes. The role of Cdk2 in hematopoietic cells and apoptosis in vivo has not yet been addressed. To determine whether Cdk2 plays a role in these cells, we performed multiple analyses of bone marrow cells, thymocytes, and splenocytes from Cdk2 knockout mice. We found that Cdk2 is not required in vivo to induce apoptosis in lymphocytes, a result that differs from previous pharmacological in vitro studies. Furthermore, thymocyte maturation was not affected by the lack of Cdk2. We then analyzed the hematopoietic stem cell compartment and found similar proportions of stem cells and progenitors in Cdk2(-)(/)(-) and wild-type animals. Knockouts of Cdk2 inhibitors (p21, p27) affect stem cell renewal, but a competitive graft experiment indicated that renewal and multilineage differentiation are normal in the absence of Cdk2. Finally, we stimulated T lymphocytes or macrophages to induce proliferation and observed normal reactivation of Cdk2(-)(/)(-) quiescent cells. Our results indicate that Cdk2 is not required for proliferation and differentiation of hematopoietic cells in vivo, although in vitro analyses consider Cdk2 to be a major player in proliferation and apoptosis in these cells and a potential target for therapy.

The scaffold protein Cybr is required for cytokine-modulated trafficking of leukocytes in vivo.

Trafficking and cell adhesion are key properties of cells of the immune system. However, the molecular pathways that control these cellular behaviors are still poorly understood. Cybr is a scaffold protein highly expressed in the hematopoietic/immune system whose physiological role is still unknown. In vitro studies have shown it regulates LFA-1, a crucial molecule in lymphocyte attachment and migration. Cybr also binds cytohesin-1, a guanine nucleotide exchange factor for the ARF GTPases, which affects actin cytoskeleton remodeling during cell migration. Here we show that expression of Cybr in vivo is differentially modulated by type 1 cytokines during lymphocyte maturation. In mice, Cybr deficiency negatively affects leukocytes circulating in blood and lymphocytes present in the lymph nodes. Moreover, in a Th1-polarized mouse model, lymphocyte trafficking is impaired by loss of Cybr, and Cybr-deficient mice with aseptic peritonitis have fewer cells than controls present in the peritoneal cavity, as well as fewer leukocytes leaving the bloodstream. Mutant mice injected with Moloney murine sarcoma/leukemia virus develop significantly larger tumors than wild-type mice and have reduced lymph node enlargement, suggesting reduced cytotoxic T-lymphocyte migration. Taken together, these data support a role for Cybr in leukocyte trafficking, especially in response to proinflammatory cytokines in stress conditions.

Synergistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2 cytokine expression.

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-gamma in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-gamma and IL-13. Analysis of the thymic subsets, CD4(-)CD8(-) (DN), CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-gamma and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-gamma(+) cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-gamma and IL-13 expression is dependent on Stat4 and NF-kappaB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-gamma. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-gamma-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.