Notch1 expression on T cells is not required for CD4+ T helper differentiation

Notch1 proteins are involved in binary cell fate decisions. To determine the role of Notch1 in the differentiation of CD4(+) Th1 versus Th2 cells, we have compared T helper polarization in vitro in naive CD4(+) T cells isolated from mice in which the N1 gene is specifically inactivated in all mature T cells. Following activation, Notch1-deficient CD4(+) T cells transcribed and secreted IFN-gamma under Th1 conditions and IL-4 under Th2 conditions at levels similar to that of control CD4(+) T cells. These results show that Notch1 is dispensable for the development of Th1 and Th2 phenotypes in vitro. The requirement for Notch1 in Th1 differentiation in vivo was analyzed following inoculation of Leishmania major in mice with a T cell-specific inactivation of the Notch1 gene. Following infection, these mice controlled parasite growth at the site of infection and healed their lesions. The mice developed a protective Th1 immune response characterized by high levels of IFN-gamma mRNA and protein and low levels of IL-4 mRNA with no IL-4 protein in their lymph node cells. Taken together, these results indicate that Notch1 is not critically involved in CD4(+) T helper 1 differentiation and in resolution of lesions following infection with L. major.     

Naturally activated CD4+ T cells are highly enriched for cytokine-producing cells

Most T cells in a normal non-immunized individual are in a resting state. However, a small proportion of splenic T cells are large activated cells both in specific pathogen-free and antigen-free mice. To further elucidate the effector functions associated with these “naturally” activated CD4⁺ T cells, we have characterized the expression of various membrane markers, cytokine production and T helper activity by these cells. We show that naturally activated CD4⁺ T cells express activation markers and contain tenfold higher proportions of cells producing IL-4, IL-10 and IFN-γ as compared to small CD4⁺ T cells. Despite the high proportion of IFN-γ producers, naturally activated CD4⁺ T cells still induce B cell proliferation and differentiation. These results are discussed in the context of normal physiological autoreactivity.     

Mini-review CD4 T cells are required for CD8 T cell memory generation

Whereas the role of CD4 T cells in B cell memory generation is well established and unequivocal, the role that CD4 T cells play in CD8 responses was until recently far more elusive and controversial. A series of recent reports, however, have re-assessed the role of CD4 help on CD8 responses and have given rise to surprisingly unambiguous conclusions. While studying very different systems, they demonstrated that CD4 T cells are absolutely required for the generation of bona fide CD8 memory cells; the reports allow, for the first time, strong analogies to be made between B and CD8 memory cell generation. These data invite us to drastically change our idea of CD4 help on CD8 responses because they show that the old dichotomy - Th-dependent versus Th-independent CD8 responses - is no longer accurate.     

T-cell immunity to murine Moloney sarcoma virus-induced tumours: L3T4+ T cells are necessary for resistance to primary sarcoma growth, but Lyt-2+ T cells are required for resistance to secondary tumour cell challenge.

Experimental protocols have been devised to deliniate the importance of T-cell subsets in immunity to Moloney sarcoma virus-induced tumours using the surface antigens L3T4 and Lyt-2 as markers of helper and cytotoxic cells, respectively. Because the monoclonal antibodies used have been shown to deplete T-cell subsets in vivo, we have been able to study the role of L3T4+ and Lyt-2+ T cells in the primary response to MSV for the first time. The results clearly show that L3T4+ T cells are the most important in resistance to the viral challenge. Mice injected with monoclonal antibodies to L3T4 grew large tumours following injection of a viral innoculum that was resisted by untreated mice or mice injected with monoclonal antibodies to Lyt-2. The same monoclonal antibodies were used to remove primed L3T4+ or Lyt-2+ T cells in vitro in adoptive transfer experiments. Normal unirradiated mice were protected from a challenge of WR19L lymphoma cells when they were given primed spleen and lymph node cells intraperitoneally. Depletion of Lyt-2+ T cells before adoptive transfer abolished this protective effect. Depletion of L3T4+ cells had no effect on the ability of primed cells to transfer immunity. Thus, while L3T4+ T cells are required for the primary rejection of MSV, only primed Lyt-2+ T cells are able to transfer resistance to a secondary challenge of lymphoma cells.     

Complement regulates CD4 T-cell help to CD8 T cells required for murine allograft rejection

Although induction of CD8 T-cell responses to transplants requires CD4-cell help, how this help is transmitted remains incompletely characterized. In vitro, cognate interactions between CD4 T cells and dendritic cells (DCs) induce C3a and C5a production. CD8(+) T cells lacking C3a receptor (C3aR) and C5a receptor (C5aR) proliferate weakly to allogeneic DCs despite CD4 help, indicating that CD4-cell help is mediated, in part, through DC-derived C3a/C5a acting on CD8(+) T cell-expressed C3aR/C5aR. In support of this concept, augmenting DC C5a/C3a production bypasses the requirement for CD4- and CD40-dependent help to wild-type CD8(+) T cells. CD4-deficient recipients of allogeneic heart transplants prime weak CD8 responses and do not acutely reject their grafts. In contrast, CD4-deficient chimeric mice possessing decay accelerating factor deficient (Daf1(-/-)) bone marrow, in which DC C3a/C5a production is potentiated, acutely reject transplants through a CD8 cell-dependent mechanism. Furthermore, hearts transplanted into CD40(-/-) mice prime weak CD8-cell responses and survive indefinitely, but hearts transplanted into Daf1(-/-)CD40(-/-) recipients undergo CD8 cell-dependent rejection. Together, the data indicate that heightened production and activation of immune cell-derived complement bypasses the need for CD40/CD154 interactions and implicate antigen-presenting cell-produced C5a and C3a as molecular bridges linking CD4 help to CD8(+) T cells.     

CD4+ T cells are required for the maintenance, not programming, of memory CD8+ T cells after acute infection

Immunization in the absence of CD4(+) T cell help results in defective CD8(+) T cell memory, deficient recall responses and diminished protective immunity. Here we investigated at what stage during the immune response to pathogen CD4(+) T cells are essential in the promotion of functional CD8(+) T cell memory. Memory CD8(+) T cell numbers decreased gradually in the absence of CD4(+) T cells despite the presence of similar numbers of memory cell precursors at the peak of the effector phase. Adoptive transfer of effector or memory CD8(+) T cells into wild-type or CD4(+) T cell-deficient mice demonstrated that the presence of CD4(+) T cells was important only after, not during, the early CD8(+) T cell programming phase. In the absence of CD4(+) T cells, memory CD8(+) T cells became functionally impaired and decreased in quantity over time. We conclude that in the context of an acute infection, CD4(+) T cells are required only during the maintenance phase of long-lived memory CD8(+) T cells.     

CD4+ T cells, but not CD8+ T cells, are required for the development of experimental autoimmune gastritis.

Murine autoimmune gastritis, induced by neonatal thymectomy, is characterized by a mononuclear infiltrate within the gastric mucosa, loss of parietal and zymogenic cells and circulating autoantibodies to the gastric H/K ATPase. The infiltrate contains both CD4+ and CD8+ T cells. Here we have investigated the roles of CD4+ and CD8+ T cells in the development of gastritis by in vivo treatment with depleting rat anti-CD4 and anti-CD8 monoclonal antibodies. Depletion of CD4+ T cells decreased the incidence of gastric mononuclear infiltrates from 63% (5/8), observed in normal rat immunoglobulin G (IgG)-injected mice, to 8% (1/12) and also abolished the production of antigastric autoantibodies. In contrast, depletion of CD8+ T cells did not reduce the incidence of gastritis. The absence of CD8+ T cells in the infiltrate of the stomach of anti-CD8(+)-treated mice was confirmed by immunocytochemistry. These results argue that neonatal thymectomy-induced autoimmune gastritis is mediated by CD4+ T cells and that CD8+ T cells do not play a significant role in the development of the gastric lesion.     

CD4+ T cells are required for antigen-specific recruitment of neutrophils by BCG-immune spleen cells.

Mycobacterium bovis bacillus Calmette-Guérin (BCG)-immune spleen cells co-inoculated into the peritoneal cavity of normal mice with BCG sonicate protein as antigen could induce an antigen-specific recruitment of neutrophils, dependent on the antigen dose and cell number. This response was significantly reduced by anti-T lymphocyte and anti-CD4 treatment of the immune spleen cells prior to the inoculation. Removal of adherent or phagocytic cells or lysis of B cells, had no significant effect. Killing of dividing cells in the splenic population induced a slight reduction in the ability of spleen cells to recruit neutrophils. M. avium sonicate protein was also able to induce BCG-immune spleen cells to mobilize neutrophils but bovine serum albumin, Listeria monocytogenes cytosolic protein and 65,000 MW heat shock protein were not. These results show that CD4+ T cells are able to induce neutrophil recruitment in an antigen-specific way during a mycobacterial infection.     

Intestinal double-positive CD4+CD8+ T cells are highly activated memory cells with an increased capacity to produce cytokines

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.     

CD4+ T cells are essential in overcoming experimental murine measles encephalitis.

Clinical observations and experimental animal models have stressed the importance of the cellular immune response in the recovery from measles virus infection. However, the relative contribution of different T-cell subsets to viral elimination is controversial. The aim of the present study was to define the components of the immune system which contribute to the control of measles virus infection. For this purpose the effect of in vivo depletion of CD4+ and/or CD8+ T lymphocytes in the murine model of experimental acute measles encephalitis was monitored with respect to disease manifestation, survival, neuropathological changes, virus elimination from brain, and antiviral antibody titre. In measles virus-resistant BALB/c mice removal of the CD8+ T-cell subset did not interfere with the clearance of virus from the brain. In contrast, depletion of CD4+ T cells rendered BALB/c mice susceptible to infection. Also, in measles virus-susceptible C3H mice CD4+ T cells played a role in recovery from measles infection, but seemed not to be as effective as CD4+ T cells from resistant BALB/c mice. The data indicate that CD4+ T cells are essential for protection against measles virus-infection of the central nervous system.     

Identification of novel markers for mouse CD4+ T follicular helper cells

CD4⁺ T follicular helper (T_FH) cells are central for generation of long‐term B‐cell immunity. A defining phenotypic attribute of T_FH cells is the expression of the chemokine R CXCR5, and T_FH cells are typically identified by co‐expression of CXCR5 together with other markers such as PD‐1, ICOS, and Bcl‐6. Herein, we report high‐level expression of the nutrient transporter folate R 4 (FR4) on T_FH cells in acute viral infection. Distinct from the expression profile of conventional T_FH markers, FR4 was highly expressed by naive CD4⁺ T cells, was downregulated after activation and subsequently re‐expressed on T_FH cells. Furthermore, FR4 expression was maintained, albeit at lower levels, on memory T_FH cells. Comparative gene expression profiling of FR4^hi versus FR4^lo Ag‐specific CD4⁺ effector T cells revealed a molecular signature consistent with T_FH and T_H1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto‐enzyme CD73, were enriched in T_FH cells compared with T_H1 cells, and phenotypic analysis confirmed expression of CD73 on T_FH cells. As there is now considerable interest in developing vaccines that would induce optimal T_FH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of T_FH cells following vaccination and infection.     

Role of CD4+ (helper) T cells in the pathogenesis of murine cytomegalovirus myocarditis.

Murine cytomegalovirus causes diffuse myocardial lesions in immunologically intact young adult male BALB/cBy mice. The cardiac changes develop in and around the small penetrating blood vessels of the heart where perivascular and interstitial infiltrates of macrophages and lymphocytes accumulate. Focal lesions of the coronary vessels and the endocardium also appear. When infected mice are depleted of CD4+ T lymphocytes, myocardial lesions fail to develop even though virus replication in the heart is enhanced. Contrary wise, when CD4+ cells are adoptively transferred into infected, thymectomized, irradiated, bone marrow-repleted mice, focal perivascular necrotizing lesions of the heart develop. Depletion of CD8+ T lymphocytes fails to influence virus replication and the development of cardiac lesions. Endothelial and endocardial cells appear to be major sites of virus replication in the heart. Delayed hypersensitivity is hypothesized to be the mechanism of cardiac injury in this model system.     

IL-36 cytokines imprint a colitogenic phenotype on CD4+ T helper cells

IL-36 cytokines are emerging as potent orchestrators of intestinal inflammation and are being implicated in the pathogenesis of inflammatory bowel diseases (IBD). However, the mechanisms through which these cytokines mediate these effects remain to be fully uncovered. Here, we report specifically elevated expression of IL-36α, and not IL-36β or IL-36γ in the serum of newly diagnosed, treatment naïve, paediatric IBD patients and identify T cells as primary cellular mediators of IL-36 responses in the inflamed gut. IL-36R expression on CD4⁺ T cells was found to promote intestinal pathology in a murine model of colitis. Consistent with these effects, IL-36R can act as a potent instructor of CD4⁺ T cell differentiation in vivo, enhancing Th1 responses, while inhibiting the generation of Tregs. In addition, loss of IL-36 responsiveness significantly reduced the migration of pathogenic CD4⁺ T cells towards intestinal tissues and IL-36 was found to act, uniquely among IL-1 family members, to induce the expression of gut homing receptors in proinflammatory murine and human CD4⁺ T cells. These data reveal an important role for IL-36 cytokines in driving the colitogenic potential of CD4⁺ T cells and identify a new mechanism through which they may contribute to disease pathogenesis.     

Increased CD4+CXCR5+T follicular helper cells in diabetic nephropathy

Background: T follicular helper (Tfh) cells are known to regulate humoral immune response. In this study we examined the correlation of different subsets of peripheral blood Tfh cells in patients with diabetic nephropathy (DN). Methods: A total of 23 DN patients and 15 healthy controls (HC) were investigated for various subsets of Tfh cells by flow cytometry. The molecules ICOS⁺, PD-1⁺, CD28⁺, CD154⁺, IL-21⁺, IFN-γ⁺, IL-4⁺, IL-17⁺ Tfh cells were examined. The subsets of B cells were investigated by flow cytometry. The levels of 24?h urinary protein and estimated glomerular filtration rate (eGFR) were calculated. A potential correlation between the number of different subsets of Tfh cells, B cells and DN, was assessed. Results: The circulating CD4⁺CXCR5⁺PD-1⁺, PD-1⁺CD154⁺, PD-1⁺CD28⁺, PD-1⁺IL-21⁺, PD-1⁺IL-4⁺, PD-1⁺-IL-17⁺-Tfh cell counts, CD38⁺CD19⁺, CD38⁺CD19⁺CD40⁺ B cells and plasma levels of IL-21 were significantly increased in DN patients (p?+CXCR5⁺PD-1⁺ Tfh cell counts negatively correlated with eGFR; Tfh cell counts positively correlated with 24?h urinary protein concentration in DN patients. Post-treatment, there was a significant reduction in the CD4⁺CXCR5⁺PD-1⁺ Tfh cell counts and its subsets, with a corresponding decrease in plasma levels of IL-6 and IL-17A (p?Conclusion: An increased number of CD4⁺CXCR5⁺PD-1⁺ Tfh cells were observed in DN patients, which may be new targets for intervention in DN.     

CD4+CD25+CD127dimFoxp3+ T cells are cytotoxic for human neurons

MS lesions are characterized by destruction of myelin and significant neuronal and axonal loss. Preliminary studies with the use of T(regs) in the mouse model of MS have been extremely encouraging. However, recent studies with human cells have shown the presence of different subpopulations of T cells within the CD4(+)CD25(+)Foxp3(+) T cell phenotype, some of which do not have regulatory functions. These findings suggest a potential difference between mouse and human in the regulatory phenotype. Here, we show that human activated CD4(+)CD25(+)Foxp3(+) T cells are neurotoxic in vitro. These cells expressed high levels of the cytotoxic molecule GrB and had no suppressive effect. On the contrary, they produced IFN-γ and low IL-17, suggesting a shift toward a T(H)1 phenotype. Thus, our data confirm the presence of a nonregulatory cytotoxic subpopulation within the human CD4(+)CD25(+)Foxp3(+) T cells and suggest further studies on the human regulatory phenotype prior to any potential therapeutic application.     

Diversion of CD4+ T cell development from regulatory T helper to effector T helper cells alters the contact hypersensitivity response

Cutaneous sensitization to reactive haptens and subsequent challenge results in a T cell-mediated response, contact hypersensitivity (CHS). Recent results from this laboratory have indicated that hapten sensitization induces two populations of reactive T cells: CD8⁺ T cells producing interferon (IFN)-γ which mediate the response and CD4⁺ T cells producing interleukin (IL)-4 and IL-10 which negatively regulate the magnitude and duration of the response. Since CD4⁺ T cell development to either IFN-γ- (Th1) or IL-4/IL-10- (Th2)-producing cells is dependent upon the cytokine environment during antigen priming, we hypothesized that CD4⁺ T cell induction in a Th1-promoting environment would not only alter the CD4⁺ T cell cytokine-producing phenotype but also the course of the CHS response. Administration of the Th1-promoting cytokine IL-12 during hapten sensitization resulted in a CHS response of greater magnitude following challenge and extended the duration of the response. In hapten-sensitized mice depleted of CD8⁺ T cells, treatment with IL-12 induced effector CD4⁺ T cells. Histological examination of challenged ear tissue from these mice indicated minimal edema and an acute mononuclear cell infiltration more typical of classical delayed-type hypersensitivity than CHS. Hapten-primed CD4⁺ T cells from IL-12 treated, sensitized mice produced IFN-γ, but not IL-4 in response to T cell receptor-mediated stimulation. Use of neutralizing anti-IFN-γ antibody indicated that IL-12 not only directly promoted Th1 development but also indirectly inhibited Th2 development through stimulation of IFN-γ production at the time of hapten sensitization. Overall, these results demonstrate that diversion of CD4⁺ T cell development to Th1 effector cells rather than to Th2 cells alters the efferent nature of CHS and removes a primary regulatory mechanism of the immune response.