Plasmodium chabaudi AS Infection Induces CD4+ Th1 Cells and Foxp3+T-bet+ Regulatory T Cells That Express CXCR3 and Migrate to CXCR3 Ligands.

Control and elimination of blood-stage Plasmodium chabaudi AS infection requires CD4+ Th1 cells that secrete IFN-γ and T follicular help (Tfh) cells together with B cell production of antibody. Foxp3+ regulatory T cells (Tregs) are also crucial to protect the host from immunopathology and severe disease, but these cells can suppress protective immune responses to malaria. The chemokine receptor CXCR3 expressed by activated T cells is important for trafficking of CD4+ Th1 cells to sites of inflammation and infection. Previous studies demonstrated CXCR3 is expressed on CD4+ T cells in the spleen during malaria, but the phenotype was not defined. We identified the phenotype of CD4+ T cells that expressed CXCR3 in C57BL/6 (B6) mice during acute P. chabaudi AS infection by analyzing expression of the transcription factors T-bet and Foxp3. We also investigated if CXCR3 contributes to control of parasite replication and survival. The frequency and number of CD4+CXCR3+ T cells increased dramatically in the spleen of infected B6 mice coincident with increased CD4+IFN-γ+ T cells. CXCR3 was up-regulated on effector CD4+Foxp3- T cells as well as Foxp3+ Tregs. Consistent with our previous observations, CD4+T-bet+Foxp3- T cells increased in B6 mice during acute infection. T-bet+Foxp3+ Tregs also increased significantly and a high frequency of these cells expressed CXCR3 supporting the notion that these cells may be Th1-like Tregs. Despite this, the percentage of CD4+Foxp3+ Tregs from infected B6 mice that migrated in vitro to the CXCR3 ligands CXCL9 and CXCL10 was significantly less than naïve mice. To investigate the in vivo contribution of CXCR3 to control of acute blood-stage malaria, we compared the course and outcome of P. chabaudi AS infection in wild-type (WT) B6 and CXCR3-deficient mice. Parasitemia levels were significantly higher around the time of peak parasitemia in CXCR3-/- compared to WT mice but survival was similar suggesting a role for CXCR3 in controlling parasite replication during acute P. chabaudi AS infection. Together, our findings indicate Th1-like CD4+T-bet+Foxp3+ Tregs that express CXCR3 are induced during acute blood-stage malaria and suggest CXCR3 expression on CD4+ Th1 cells may contribute to their migration to the spleen.

Regulating the adaptive immune response to blood-stage malaria: role of dendritic cells and CD4⁺Foxp3⁺ regulatory T cells.

Although a clearer understanding of the underlying mechanisms involved in protection and immunopathology during blood-stage malaria has emerged, the mechanisms involved in regulating the adaptive immune response especially those required to maintain a balance between beneficial and deleterious responses remain unclear. Recent evidence suggests the importance of CD11c⁺ dendritic cells (DC) and CD4⁺Foxp3⁺ regulatory T cells in regulating immune responses during infection and autoimmune disease, but information concerning the contribution of these cells to regulating immunity to malaria is limited. Here, we review recent findings from our laboratory and others in experimental models of malaria in mice and in Plasmodium-infected humans on the roles of DC and natural regulatory T cells in regulating adaptive immunity to blood-stage malaria.

IL-2 contributes to maintaining a balance between CD4+Foxp3+ regulatory T cells and effector CD4+ T cells required for immune control of blood-stage malaria infection.

To investigate the role of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells in blood-stage malaria, we compared Plasmodium chabaudi AS infection in wild-type (WT) C57BL/6 and transgenic mice overexpressing the transcription factor Foxp3 (Foxp3Tg) and observed that Foxp3Tg mice experienced lethal infection and deficient malaria-specific immune responses. Adoptive transfer of total CD4(+) T cells from Foxp3Tg mice or CD4(+)CD25(+) T cells from WT mice to naive WT recipients confirmed that high numbers of Treg cells compromised immune control of malaria. Transfer of GFP(+)CD4(+)CD25(+) T cells to naive WT recipients together with immunohistochemical staining of spleens from infected WT mice demonstrated that Foxp3(+) Treg cells localized in the T cell area of the spleen. Determination of CD4(+)Foxp3(+) Treg cell responses in the spleen of infected WT mice revealed a significant but transient increase in CD4(+)Foxp3(+) Treg cells early in infection. This was followed by a significant and sustained decrease due to reduced proliferation and apoptosis of CD4(+)Foxp3(+) Treg cells. Importantly, the kinetics of IL-2 secretion by effector CD4(+)Foxp3(-) T cells coincided with changes in CD4(+)Foxp3(+) cells and the differentiation of CD4(+)T-bet(+)IFN-γ(+) cells required for immune control of infection. Administration of the IL-2/anti-IL-2 mAb (clone JES6-1) complex to infected WT mice increased the severity of P. chabaudi AS infection and promoted expansion of Foxp3(+) Treg cells. Collectively, these data demonstrate that the ability to control and eliminate P. chabaudi AS infection is due to a tight balance between natural Treg cells and effector CD4(+) Th1 cells, a balance regulated in part by IL-2.

Role of high-affinity HLA-DP specific CLIP-derived peptides in beryllium binding to the HLA-DPGlu69 berylliosis-associated molecules and presentation to beryllium-sensitized T cells.

Berylliosis is driven by the accumulation in the lung of beryllium-specific T helper type 1 (Th1) cells recognizing beryllium as antigen when presented principally by human leucocyte antigen DP molecules carrying a glutamate at position beta69 (HLA-DPGlu69). This study was designed to clarify the precise role of peptides in beryllium binding to the HLA-DP groove's pocket 4 and to identify peptides with higher affinity for pocket 4 that might prevent beryllium presentation and T-cell stimulation. Beryllium/HLA-DP interactions were analysed by the ability of beryllium to compete with CLIP and CLIP-derived peptides to HLA-DPGlu69 soluble molecule. The CLIP-derived low-affinity peptide CLIP-AA, could not outcompete beryllium; while the CLIP-derived high-affinity peptides CLIP-YY, CLIP-QY and CLIP-RF were only marginally influenced by the presence of beryllium in the competition assay. The effect of these CLIP-derived high-affinity peptides on beryllium presentation was determined by measuring interferon-gamma (IFN-gamma) release upon beryllium stimulation of peripheral blood mononuclear cells obtained from beryllium-hypersensitive subjects. CLIP-YY did inhibit beryllium presentation and T-cell activation, while CLIP-QY and CLIP-RF markedly enhanced the IFN-gamma response to beryllium. Anti-HLA-DP monoclonal antibody blocked the beryllium-induced IFN-gamma release in the presence of CLIP-QY (88%) and CLIP-RF (76%). A similar effect was observed for CLIP-YY capability to block IFN-gamma release by beryllium stimulation in the presence of CLIP-QY (79%) and CLIP-RF (76%). Overall, these data support the proposal that HLA-DP high-affinity peptides might be used as a model for specific berylliosis therapy.

[Berylliosis in Italy: a case of "sarcoidosis" under the threshold limit value]. / La berilliosi in Italia: una caso di "sarcoidosi" sotto la soglia di esposizione.

BACKGROUND: Berylliosis is caused by a chronic immune reaction to beryllium; in Italy the first case of beryllium exposure-related disease was described in 1935 by Fabroni-Marradi and two additional cases of beryllium disease were subsequently described by Ambrosi and co-workers in 1968. No case has since been recognized using the standardized criteria including immunological testing. OBJECTIVES: To describe a case report of clinically significant berylliosis that occurred in a man exposed to beryllium for ten years in the workplace at concentrations below the permitted threshold limit value. METHODS: The man complained of dyspnoea, dry cough, weakness and weight loss for the past year and was at first diagnosed as suffering from sarcoidosis because of increased angiotensin converting enzyme levels, alteration of hepatic and renal functional indexes, the presence of diffused reticulo-nodular lung abnormalities with high resolution computed tomography that also showed enlarged mediastinal lymph nodes, abnormal lung physiology with reduced diffusion capacity and a bronchial biopsy showing granulomatous lesions. Because of the occupational history immunological testing and high resolution HLA class II typing were performed. RESULTS: The high response to beryllium in the lymphocytes proliferation test and the HLA typing which revealed the presence of the two susceptibility markers HLA-DPGlu69 and HLA-DRPhe47 led to a diagnosis of berylliosis. CONCLUSIONS: The importance is stressed of suspecting a diagnosis of berylliosis in the proper occupational contexts and encouraging the use of immunological tests for diagnosis, and also the need for critical revision of the permitted threshold limit values.

Identification of HLA-DRPhebeta47 as the susceptibility marker of hypersensitivity to beryllium in individuals lacking the berylliosis-associated supratypic marker HLA-DPGlubeta69.

BACKGROUND: Susceptibility to beryllium (Be)-hypersensitivity (BH) has been associated with HLA-DP alleles carrying a glutamate at position 69 of the HLA-DP beta-chain (HLA-DPGlu69) and with several HLA-DP, -DQ and -DR alleles and polymorphisms. However, no genetic associations have been found between BH affected subjects not carrying the HLA-DPGlu69 susceptibility marker. METHODS: In this report, we re-evaluated an already described patient populations after 7 years of follow-up including new 29 identified BH subjects. An overall population 36 berylliosis patients and 38 Be-sensitization without lung granulomas and 86 Be-exposed controls was analysed to assess the role of the individual HLA-class II polymorphisms associated with BH-susceptibility in HLA-DPGlu69 negative subjects by univariate and multivariate analysis. RESULTS: As previously observed in this population the HLA-DPGlu69 markers was present in higher frequency in berylliosis patients (31 out of 36, 86%) than in Be-sensitized (21 out of 38, 55%, p = 0.008 vs berylliosis) and 41 out of 86 (48%, p < 0.0001 vs berylliosis, p = 0.55 vs Be-sensitized) Be-exposed controls.However, 22 subjects presenting BH did not carry the HLA-DPGlu69 marker. We thus evaluated the contribution of all the HLA-DR, -DP and -DQ polymorphisms in determining BH susceptibility in this subgroup of HLA-Glu69 subjects. In HLA-DPGlu69-negatives a significant association with BH was found for the HLA-DQLeu26, for the HLA-DRB1 locus residues Ser13, Tyr26, His32, Asn37, Phe47 and Arg74 and for the HLA-DRB3 locus clusterized residues Arg11, Tyr26, Asp28, Leu38, Ser60 and Arg74. HLA-DRPhe47 (OR 2.956, p < 0.05) resulting independently associated with BH. Further, Be-stimulated T-cell proliferation in the HLA-DPGlu69-negative subjects (all carrying HLA-DRPhe47) was inhibited by the anti-HLA-DR antibody (range 70-92% inhibition) significantly more than by the anti-HLA-DP antibody (range: 6-29%; p < 0.02 compared to anti-HLA-DR) while it was not affected by the anti-HLA-DQ antibody. CONCLUSION: We conclude that HLA-DPGlu69 is the primary marker of Be-hypersensitivity and HLA-DRPhe47 is associated with BH in Glu69-negative subjects, likely playing a role in Be-presentation and sensitization.