Identification of an expanded population of activated CD4(+) CD25(+) T cells expressing CD45RO and IL-7R in kidney transplant recipients

Recent studies have shown that CD4+ CD25+ T cells belong to two functionally different T lymphocytes, i.e. regulatory T cells (Treg) or activated T cells (Tact), which can be distinguished based on the expression of CD45RO and IL-7R: Treg (FoxP3+) are CD45RO+ IL-7R- , whereas Tact (FoxP3- ) are CD45RO+ IL- 7R+. In order to determine if a CD4+ CD25+ CD45RO+ IL-7R+ activated T cell population might be identified in kidney transplant recipients, we studied 27 healthy subjects (HS) and 23 kidney recipients, of whom 17 had stable graft function under standard immunosuppression (IS), 5 had biopsy-proven chronic humoral rejection (CHR), and one was a stable "tolerant" patient who had discontinued IS for more than 2 years. Phenotypical analysis by flow cytometry and functional assays by MLR were performed. Overall, the Tact population was found to be significantly increased in 87% of the transplant recipients (mean: 18.8±10.1% of CD4+ CD25+ T cells) compared to HS (mean: 4.5±2.0%; P<0.0001). In the 5 patients with CHR, this Tact population was highly expanded (31.3±9.3%; P<0.0001), whereas it was comparable to HS in the "tolerant" recipient (4.7%). Intermediate levels (16.0±6.9%; P<0.0001) were found in the 17 stable recipients. In CHR, the proliferative capacity of the Tact population was found to be 5-fold higher when stimulated by irradiated donor PBMC as compared to a stimulation by irradiated 3rd party PBMC. After kidney transplantation, an expanded circulating CD4+ CD25+ T cell population characterized by the expression of CD45RO and IL-7R was found in most recipients, particularly in those with CHR. In a patient with long-term operational tolerance, this Tact population was similar to HS. Measuring circulating Tact may become a useful monitoring tool after transplantation.

Different epitopes of the LACK protein are recognized by V beta 4 V alpha 8 CD4+ T cells in H-2b and H-2d mice susceptible to Leishmania major.

After inoculation of Leishmania major, a rapid production of IL-4 by LACK-specific CD4+ T cells has been shown to drive Th2 cell development in susceptible mice i.e. BALB/c and C57BL/6 mice rendered susceptible by neutralization of IFN-gamma at the onset of infection. Here, we showed that peptide AA 156-173 induced an early IL-4 mRNA expression not only in BALB/c mice but also in resistant B10.D2 mice when IFN-gamma is neutralized. Epitope mapping of LACK protein demonstrated that peptide containing AA 293-305 induced early IL-4 mRNA transcripts in susceptible H-2b mice i.e. BALB/b and resistant C57BL/6 mice when IFN-gamma is neutralized. Stringently, the early IL-4 response to the H-2d (AA 156-173) or the H-2b (AA 293-305) epitopes occurred in V beta 4 V alpha 8 CD4+ T cells from either H-2d or H-2b susceptible mice, respectively.

Differential requirement for CD4 help in the development of an antigen-specific CD8+ T cell response depending on the route of immunization.

Previous studies in our laboratory have shown that DBA/2 mice injected i.p. with syngeneic P815 tumor cells transfected with the HLA-CW3 gene (P815-CW3) showed a dramatic expansion of activated CD8+CD62L- T cells expressing exclusively the Vbeta10 segment. We have used this model to study the regulatory mechanisms involved in the development of the CW3-specific CD8+ response, with respect to different routes of immunization. Whereas both intradermal (i.d.) and i.p. immunization of DBA/2 mice with P815-CW3 cells led to a strong expansion of CD8+CD62L-Vbeta10+ cells, only the i.d. route allowed this expansion after immunization with P815 cells transfected with a minigene coding for the antigenic epitope CW3 170-179 (P815 miniCW3). Furthermore, depletion of CD4+ T cells in vivo completely abolished the specific response of CD8+CD62L-Vbeta10+ cells and prevented the rejection of P815-CW3 tumor cells injected i.p., whereas it did not affect CD8S+CD62L-Vbeta10+ cell expansion after i.d. immunization with either P815-CW3 or P815 miniCW3. Finally, the CW3-specific CD8+ memory response was identical whether or not CD4+ T cells were depleted during the primary response. Collectively, these results suggest that the CD8+ T cell response to P815-CW3 tumor cells injected i.p. is strictly dependent upon recognition of a helper epitope by CD4+ T cells, whereas no such requirement is observed for i.d. injection.

CD4+ T cell count recovery in HIV type 1-infected patients is independent of class of antiretroviral therapy.

BACKGROUND: In recent years, treatment options for human immunodeficiency virus type 1 (HIV-1) infection have changed from nonboosted protease inhibitors (PIs) to nonnucleoside reverse-transcriptase inhibitors (NNRTIs) and boosted PI-based antiretroviral drug regimens, but the impact on immunological recovery remains uncertain. METHODS: During January 1996 through December 2004 [corrected] all patients in the Swiss HIV Cohort were included if they received the first combination antiretroviral therapy (cART) and had known baseline CD4(+) T cell counts and HIV-1 RNA values (n = 3293). For follow-up, we used the Swiss HIV Cohort Study database update of May 2007 [corrected] The mean (+/-SD) duration of follow-up was 26.8 +/- 20.5 months. The follow-up time was limited to the duration of the first cART. CD4(+) T cell recovery was analyzed in 3 different treatment groups: nonboosted PI, NNRTI, or boosted PI. The end point was the absolute increase of CD4(+) T cell count in the 3 treatment groups after the initiation of cART. RESULTS: Two thousand five hundred ninety individuals (78.7%) initiated a nonboosted-PI regimen, 452 (13.7%) initiated an NNRTI regimen, and 251 (7.6%) initiated a boosted-PI regimen. Absolute CD4(+) T cell count increases at 48 months were as follows: in the nonboosted-PI group, from 210 to 520 cells/muL; in the NNRTI group, from 220 to 475 cells/muL; and in the boosted-PI group, from 168 to 511 cells/muL. In a multivariate analysis, the treatment group did not affect the response of CD4(+) T cells; however, increased age, pretreatment with nucleoside reverse-transcriptase inhibitors, serological tests positive for hepatitis C virus, Centers for Disease Control and Prevention stage C infection, lower baseline CD4(+) T cell count, and lower baseline HIV-1 RNA level were risk factors for smaller increases in CD4(+) T cell count. CONCLUSION: CD4(+) T cell recovery was similar in patients receiving nonboosted PI-, NNRTI-, and boosted PI-based cART.

Deregulated MHC class II transactivator expression leads to a strong Th2 bias in CD4+ T lymphocytes.

The MHC class II (MHC-II) transactivator (CIITA) is the master transcriptional regulator of genes involved in MHC-II-restricted Ag presentation. Fine tuning of CIITA gene expression determines the cell type-specific expression of MHC-II genes. This regulation is achieved by the selective usage of multiple CIITA promoters. It has recently been suggested that CIITA also contributes to Th cell differentiation by suppressing IL-4 expression in Th1 cells. In this study, we show that endogenous CIITA is expressed at low levels in activated mouse T cells. Importantly CIITA is not regulated differentially in murine and human Th1 and Th2 cells. Ectopic expression of a CIITA transgene in multiple mouse cell types including T cells, does not interfere with normal development of CD4(+) T cells. However, upon TCR activation the CIITA transgenic CD4(+) T cells preferentially differentiate into IL-4-secreting Th2-type cells. These results imply that CIITA is not a direct Th1-specific repressor of the IL-4 gene and that tight control over the expression of CIITA and MHC-II is required to maintain the normal balance between Th1 and Th2 responses.

Anti-leishmania effector functions of CD4+ Th1 cells and early events instructing Th2 cell development and susceptibility to Leishmania major in BALB/c mice.

Resistance and susceptibility to infection with the intracellular parasite, Leishmania major, are mediated by parasite-specific CD4+ Th1 and Th2 cells, respectively. It is well established that the protective effect of parasite-specific CD4+ Th1 cells is largely dependent upon the IFN-gamma produced. However, recent results indicate that the effect of Th1 cells on resolution of lesions induced by L. major in genetically resistant mice also requires a functional Fas-FasL pathway of cytotoxicity. In contrast to resistant mice, susceptible BALB/c mice develop aberrant Th2 responses following infection with L. major and consequently suffer progressive disease. These outcomes clearly depends upon the production of interleukin 4 (IL-4) early after infection. We have shown that a burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hrs after infection, occurs within CD4+ T cells that express V beta 4-V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and Th1 responses occurred following infection. The LACK antigen of L. major induced comparable IL-4 production in V beta 4-V alpha 8 CD4+ T cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4-V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex parasite. The IL-4 produced rapidly by these CD4+ T cells induces within 48 hours a state of unresponsiveness to IL-12 among parasite-specific CD4+ T cell precursors by downregulating the IL-12 receptor beta 2 chain expression.

CD4 T cells producing pro-inflammatory interleukin-17 mediate high pathology in schistosomiasis

In murine schistosomiasis mansoni, pronounced CD4 T cell-mediated, egg-induced, hepato-intestinal immunopathology and death, whether genetically determined or elicited experimentally, are associated with failure to down-regulate a net pro-inflammatory immune response. Important evidence contributing to this notion comes from the observation that immunization with schistosome egg antigens in CFA (SEA/CFA) causes low pathology C57BL/6 mice to develop an exacerbated form of disease and death in a cytokine milieu characterized by elevated interferon (IFN)-gamma levels. Since such a pro-inflammatory environment presumes a signaling pathway involving interleukin (IL)-12, the SEA/CFA immunization model was used to examine the extent of hepatic immunopathology in the absence of this cytokine. Surprisingly, the IL-12p40 subunit was an absolute requirement for the development of exacerbated disease, whereas the IL-12p35 subunit was not. Moreover, significantly elevated in vitro production of IL-17, but not of IFN-gamma, correlated with the high pathology, and neutralization of IL-17 in vivo resulted in a significant reduction of hepatic inflammation. Our findings clearly demonstrate the pathogenic potential of the novel IL-17-producing T cell subpopulation (ThIL-17), previously shown to mediate chronic inflammation in autoimmune disease. They also imply that IL-23, but not IL-12, is the critical signal necessary to support the pro-inflammatory ThIL-17 subset involved in high pathology schistosomiasis.

Combined Use of Mycobacterium tuberculosis-Specific CD4 and CD8 T-Cell Responses Is a Powerful Diagnostic Tool of Active Tuberculosis.

Immune-based assays are promising tools to help to formulate diagnosis of active tuberculosis. A multiparameter flow cytometry assay assessing T-cell responses specific to Mycobacterium tuberculosis and the combination of both CD4 and CD8 T-cell responses accurately discriminated between active tuberculosis and latent infection.

The role of triple infection with hepatitis B virus, hepatitis C virus, and human immunodeficiency virus (HIV) type-1 on CD4+ lymphocyte levels in the highly HIV infected population of North-Central Nigeria

We set out to determine the seroprevalence of hepatitis B and C among human immunodeficiency virus type-1 (HIV-1) infected individuals in North-Central Nigeria to define the influence of these infections on CD4+ lymphocytes cells among our patients as access to antiretroviral therapy improves across the Nigerian nation. The CD4+ values of 180 confirmed HIV-1 infected individuals were enumerated using a superior fluorescence-activated cell sorter system. These patients were tested for the presence of hepatitis B surface antigen and anti-hepatitis C virus (HCV) using third generation enzyme-linked immunosorbent assays. Fifty (27.8%) patients had active hepatitis B virus (HBV) infection while 33 (18.3%) tested positive for anti-HCV antibody. Of these infections, 110 (61.1%), 37 (20.6%), and 20 (11.1%) had HIV only, HBV/HIV-only, and HCV/HIV-only respectively. A HBV/HCV/HIV coinfection prevalence of 7.2% (13 patients) was recorded. Patients coinfected with HIV/HBV/HCV appeared to have lower CD4+ counts (mean = 107 cells/µl; AIDS defining) when compared to HBV/HIV-only (mean = 377 cells/µl), HCV/HIV-only (mean = 373 cells/µl) and patients with mono HIV infection (mean = 478 cells/µl). Coinfection with HBV or HCV is relatively common among HIV-infected patients in Nigeria and should be a big consideration in the initiation and choice of therapy.

IL-4 rapidly produced by V beta 4 V alpha 8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice.

BALB/c mice develop aberrant T helper 2 (Th2) responses and suffer progressive disease after infection with Leishmania major. These outcomes depend on the production of interleukin-4 (IL-4) early after infection. Here we demonstrate that the burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hr after infection, occurs within CD4+ T cells that express V beta 4 V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient BALB/c mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and T helper 1 responses occurred following infection. Recombinant LACK antigen from L. major induced comparable IL-4 production in V beta 4 V alpha 8 CD4+ cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4 V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex organism.

Biomedical nanoparticles modulate specific CD4(+) T cell stimulation by inhibition of antigen processing in dendritic cells

Understanding how nanoparticles may affect immune responses is an essential prerequisite to developing novel clinical applications. To investigate nanoparticle-dependent outcomes on immune responses, dendritic cells (DCs) were treated with model biomedical poly(vinylalcohol)-coated super-paramagnetic iron oxide nanoparticles (PVA-SPIONs). PVA-SPIONs uptake by human monocyte-derived DCs (MDDCs) was analyzed by flow cytometry (FACS) and advanced imaging techniques. Viability, activation, function, and stimulatory capacity of MDDCs were assessed by FACS and an in vitro CD4(+) T cell assay. PVA-SPION uptake was dose-dependent, decreased by lipopolysaccharide (LPS)-induced MDDC maturation at higher particle concentrations, and was inhibited by cytochalasin D pre-treatment. PVA-SPIONs did not alter surface marker expression (CD80, CD83, CD86, myeloid/plasmacytoid DC markers) or antigen-uptake, but decreased the capacity of MDDCs to process antigen, stimulate CD4(+) T cells, and induce cytokines. The decreased antigen processing and CD4(+) T cell stimulation capability of MDDCs following PVA-SPION treatment suggests that MDDCs may revert to a more functionally immature state following particle exposure.

MAGE-A3 and MAGE-A4 specific CD4(+) T cells in head and neck cancer patients: detection of naturally acquired responses and identification of new epitopes.

Frequent expression of cancer testis antigens (CTA) has been consistently observed in head and neck squamous cell carcinomas (HNSCC). For instance, in 52 HNSCC patients, MAGE-A3 and -A4 CTA were expressed in over 75% of tumors, regardless of the sites of primary tumors such as oral cavity or hypopharynx. Yet, T-cell responses against these CTA in tumor-bearing patients have not been investigated in detail. In this study, we assessed the naturally acquired T-cell response against MAGE-A3 and -A4 in nonvaccinated HNSCC patients. Autologous antigen-presenting cells pulsed with overlapping peptide pools were used to detect and isolate MAGE-A3 and MAGE-A4 specific CD4(+) T cells from healthy donors and seven head and neck cancer patients. CD4(+) T-cell clones were characterized by cytokine secretion. We could detect and isolate MAGE-A3 and MAGE-A4 specific CD4(+) T cells from 7/7 cancer patients analyzed. Moreover, we identified six previously described and three new epitopes for MAGE-A3. Among them, the MAGE-A3(111-125) and MAGE-A3(161-175) epitopes were shown to be naturally processed and presented by DC in association with HLA-DP and DR, respectively. All of the detected MAGE-A4 responses were specific for new helper epitopes. These data suggest that naturally acquired CD4(+) T-cell responses against CT antigens often occur in vivo in HNSCC cancer patients and provide a rationale for the development of active immunotherapeutic approaches in this type of tumor.

MicroRNA Profile of Circulating CD4-positive Regulatory T Cells in Human Adults and Impact of Differentially Expressed MicroRNAs on Expression of Two Genes Essential to Their Function.

Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4(+) Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3'-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3'-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4(+) adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3'-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4(+) Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.

Postpartum changes in plasma viral load and CD4 percentage among HIV-infected women from Latin American and Caribbean countries: the NISDI Perinatal Study

The goal of this study was to evaluate changes in plasma human immunodeficiency virus (HIV) RNA concentration [viral load (VL)] and CD4+ percentage (CD4%) during 6-12 weeks postpartum (PP) among HIV-infected women and to assess differences according to the reason for receipt of antiretrovirals (ARVs) during pregnancy [prophylaxis (PR) vs. treatment (TR)]. Data from a prospective cohort of HIV-infected pregnant women (National Institute of Child Health and Human Development International Site Development Initiative Perinatal Study) were analyzed. Women experiencing their first pregnancy who received ARVs for PR (started during pregnancy, stopped PP) or for TR (initiated prior to pregnancy and/or continued PP) were included and were followed PP. Increases in plasma VL (> 0.5 log10) and decreases in CD4% (> 20% relative decrease in CD4%) between hospital discharge (HD) and PP were assessed. Of the 1,229 women enrolled, 1,119 met the inclusion criteria (PR: 601; TR: 518). At enrollment, 87% were asymptomatic. The median CD4% values were: HD [34% (PR); 25% (TR)] and PP [29% (PR); 24% (TR)]. The VL increases were 60% (PR) and 19% (TR) (p < 0.0001). The CD4% decreases were 36% (PR) and 18% (TR) (p < 0.0001). Women receiving PR were more likely to exhibit an increase in VL [adjusted odds ratio (AOR) 7.7 (95% CI: 5.5-10.9) and a CD4% decrease (AOR 2.3; 95% CI: 1.6-3.2). Women receiving PR are more likely to have VL increases and CD4% decreases compared to those receiving TR. The clinical implications of these VL and CD4% changes remain to be explored.

Toward Protein Biomarkers for Allergy: CD4+ T Cell Proteomics in Allergic and Nonallergic Subjects Sampled in and out of Pollen Season.

Allergy is an immunological disorder of the upper airways, lung, skin, and the gut with a growing prevalence over the last decades in Western countries. Atopy, the genetic predisposition for allergy, is strongly dependent on familial inheritance and environmental factors. These observations call for predictive markers of progression from atopy to allergy, a prerequisite to any active intervention in neonates and children (prophylactic interventions/primary prevention) or in adults (immunomodulatory interventions/secondary prevention). In an attempt to identify early biomarkers of the "atopic march" using minimally invasive sampling, CD4+ T cells from 20 adult volunteers (10 healthy and 10 with respiratory allergies) were isolated and quantitatively analyzed and their proteomes were compared in and out of pollen season (± antigen exposure). The proteome study based on high-resolution 2D gel electrophoresis revealed three candidate protein markers that distinguish the CD4+ T cell proteomes of normal from allergic individuals when sampled out of pollen season, namely Talin 1, Nipsnap homologue 3A, and Glutamate-cysteine ligase regulatory protein. Three proteins were found differentially expressed between the CD4+ T cell proteomes of normal and allergic subjects when sampled during pollen season: carbonyl reductase, glutathione S-transferase ω 1, and 2,4-dienoyl-CoA reductase. The results were partly validated by Western blotting.