The role of cytokines in T-cell memory in health and disease

Upon stimulation with their cognate antigen, naive T cells undergo proliferation and differentiation into effector cells, followed by apoptosis or survival as precursors of long-lived memory cells. These phases of a T-cell response and the ensuing maintenance of memory T cells are shaped by cytokines, most notably interleukin-2 (IL-2), IL-7, and IL-15 that share the common γ chain (γ_c) cytokine receptor. Steady-state production of IL-7 and IL-15 is necessary for background proliferation and homeostatic survival of CD4⁺ and CD8⁺ memory T cells. During immune responses, augmented levels of IL-2, IL-15, IL-21, IL-12, IL-18, and type-I interferons determine the memory potential of antigen-specific effector CD8⁺ cells, while increased IL-2 and IL-15 cause bystander proliferation of heterologous CD4⁺ and CD8⁺ memory T cells. Limiting availability of γ_c cytokines, reduction in regulatory T cells or IL-10, and persistence of inflammation or cognate antigen can result in memory T cells, which fail to become cytokine-dependent long-lived cells. Conversely, increased IL-7 and IL-15 can expand memory T cells, including pathogenic tissue-resident memory T cells, as seen in lymphopenia and certain chronic-inflammatory disorders and malignancies. These abovementioned factors impact immunotherapy and vaccines directed at memory T cells in cancer and chronic infection.     

Quality assurance of intracellular cytokine staining assays: Analysis of multiple rounds of proficiency testing

When evaluating candidate prophylactic HIV and cancer vaccines, intracellular cytokine staining (ICS) assays that measure the frequency and magnitude of antigen-specific T-cell subsets are one tool to monitor immunogen performance and make product advancement decisions. To assess the inter-laboratory assay variation among multiple laboratories testing vaccine candidates, the NIH/NIAID/DAIDS in collaboration with BD Biosciences implemented an ICS Quality Assurance Program (QAP). Seven rounds of testing have been conducted in which 16 laboratories worldwide participated. In each round, IFN-γ, IL-2 and/or TNF-α responses in CD4+ and CD8+ T-cells to CEF or CMV pp65 peptide mixes were tested using cryopreserved peripheral blood mononuclear cells (PBMC) from CMV seropositive donors. We found that for responses measured above 0.2%, inter-laboratory %CVs were, on average, 35%. No differences in inter-laboratory variation were observed if a 4-color antibody cocktail or a 7-color combination was used. Moreover, the data allowed identification of important sources of variability for flow cytometry-based assays, including: number of collected events, gating strategy and instrument setup and performance. As a consequence, in this multi-site study we were able to define pass and fail criteria for ICS assays, which will be adopted in the subsequent rounds of testing and could be easily extrapolated to QAP for other flow cytometry-based assays.     

Optimization of intracellular cytokine staining for the quantitation of antigen-specific CD4+ T cell responses in rhesus macaques

Standard proliferation assays used for analysis of CD4+ T cell function have significant shortcomings, including limited sensitivity, lack of truly quantitative readouts and significant variability. We have optimized an intracellular cytokine staining (ICS) assay in rhesus macaques which allows us to identify virus-specific CD4+ T cells at the single-cell level with high sensitivity while reducing background staining to a minimum. A variety of parameters were tested to determine the optimal experimental conditions necessary for the detection of antigen-specific CD4+ T cells in macaques. Central to our optimized protocol was the addition of cross-linked costimulatory anti-CD28 and anti-CD49d Mabs, a modification which resulted in up to threefold enhancement of the frequency of TNF-alpha-secreting CD4+ T cells following superantigen- or antigen-specific stimulation. The ICS protocol was also optimized with respect to antigen concentration and duration of antigenic stimulation. These modifications resulted in a convenient and highly reproducible assay with intra- and inter-assay variability of less than 10%. Although cryopreservation of PBMC generally led to a 40% to 80% decrease in the frequency of antigen-specific CD4+ T cells detected by ICS using stimulation with viral proteins, the use of overlapping peptide pools minimized the effects of cryopreservation on ICS responses. The use of more sensitive techniques such as ICS permits delineation of antigen-specific cells at the single cell level and should provide new insights into pathogen-specific immune responses in the rhesus macaque model.     

Development of a whole blood intracellular cytokine staining assay for mapping CD4(+) and CD8(+) T-cell responses across the HIV-1 genome

A whole blood peptide mapping intracellular cytokine staining (ICS) assay was developed that allows the direct comparison, at the individual peptide level, of CD4(+) and CD8(+) T-cell responses that span every encoded protein, in patients infected with HIV-1. Whole blood samples from HIV-1 infected patients were stimulated with overlapping synthetic peptides spanning nine subtype C HIV-1 gene regions (Gag, Pol, Nef, Env, Tat, Rev, Vif, Vpu, Vpr). Following stimulation and permeabilization, cells were stained with fluorochrome labelled antibodies to CD3, CD8 (CD4(+) cells were defined as CD8 negative cells), and IL-2 and IFN-gamma. A total of 396 overlapping peptides were arranged in pools with a matrix design which allowed the identification of individual peptide responses from multiple pool responses. HIV-1 infected patients screened using this method showed a broad range of peptide responses across the entire HIV-1 genome with CD8 T-cell responses being higher in frequency in magnitude than CD4(+) T-cell responses. The advantages of this whole blood ICS assay include the following: (1) the response to all potential HIV-1 epitopes across the genome can be examined, (2) the responding cell type can be monitored in the same reaction, and (3) considerably less blood is required than would be necessary if peripheral blood mononuclear cells (PBMC) were first isolated prior to peptide stimulation.     

Frontline: An in-depth evaluation of the production of IL-2 by antigen-specific CD8 T cells in vivo

IL-2 is an important cytokine that is capable of inducing both proliferation and apoptosis of activated T cells. CD4 T cells are thought to be the major producers of IL-2, but CD8 T cells also produce copious amounts of this cytokine. However, our current understanding regarding the kinetics of IL-2 production by antigen-specific CD8 T cells, and the proportion of these cells that produce IL-2 in vivo, is extremely limited. We now demonstrate that virus-specific CD8 T cells initiate IL-2 production by 6 h post-infection and prior to cell division in vivo. Interestingly, peak levels of IL-2 production were achieved very early during the response and prior to the proliferative peak. We also show -- using transgenic mice expressing herpes simplex virus-1 thymidine kinase under the control of the IL-2 promoter -- that, unlike what has been reported for antigen-specific CD4 T cells, the majority of antigen-specific CD8 T cells produce IL-2 during primary as well as secondary immune responses in vivo.     

Enhanced ELISPOT detection of antigen-specific T cell responses from cryopreserved specimens with addition of both IL-7 and IL-15--the Amplispot assay

The importance of the enzyme-linked immunosorbent spot (ELISPOT) assay as a tool for studying immune responses in vitro is becoming increasingly apparent. However, there remains a need for enhanced sensitivity for the detection of low frequency antigen-specific T cell responses. We reasoned that the addition of a combination of the cytokines interleukin (IL)-7 and IL-15 would selectively increase interferon-gamma (IFN-gamma) production from antigen-stimulated CD4+ and CD8+ effector memory T cells. Freshly isolated or cryopreserved peripheral blood mononuclear cells (PBMC) from four healthy donors were analysed by ELISPOT for the frequency of purified protein derivative (PPD)-specific CD4+ T cells or cytomegalovirus (CMV) peptide-specific CD8+ T cells. Addition of IL-7 and IL-15 increased the number of PPD-specific CD4+ T cells up to 2.4-fold in fresh PBMC and up to 18-fold in cryopreserved PBMC. The cytokines also increased the number of CMV peptide-specific CD8+ T cells in fresh PBMC up to 7.5-fold. No additional increases were seen when antibodies to co-stimulatory molecules CD28 and CD49d were applied together with the cytokine combination. These data demonstrate that the sensitivity of the ELISPOT assay may be significantly augmented by addition of the cytokines IL-7 and IL-15 to antigen-stimulated cells. This method will be particularly useful for the assessment of antigen-stimulated cytokine production by T cells in cryopreserved biological specimens.     

使用活化诱导标记法评价HIV-1感染者特异性CD4+T细胞免疫应答的研究

目的:建立一种检测HIV-1特异性CD4⁺T细胞亚群功能的活化诱导标记法(activation-induced markers,AIM),从而更有效地评价HIV-1抗原特异性CD4⁺T细胞免疫应答水平。方法:选取12例未经抗病毒治疗的HIV-1慢性感染者及6例未感染HIV-1的健康人,分别以基于多色流式细胞术的AIM法和细胞内因子染色法(intracellular cytokine staining,ICS)检测抗原特异性T淋巴细胞功能,并探讨两种方法用于评价HIV-1感染者抗原特异性T细胞免疫应答的能力。结果:AIM法检测HIV-1慢性感染者中HIV-1抗原特异性PD-1⁺CD25⁺CD4⁺T、CD69⁺CD200⁺CD4⁺T、CD69⁺ICOS⁺CD4⁺T细胞阳性的比例为11/12、8/12和7/12,检测CD69⁺ICOS⁺CD8⁺T、CD137⁺CD69⁺CD8⁺T、PD-1⁺CD25⁺CD8⁺、OX40⁺PD-1⁺CD8⁺T细胞阳性的比例为8/12、8/12、7/12、7/12。ICS法检测HIV-1抗原特异性IL-2⁺CD4⁺T、IFN-γ⁺CD4⁺T、TNF-α⁺CD4⁺T细胞阳性的占比为2/12、2/12、0;IFN-γ⁺CD8⁺T、TNF-α⁺CD8⁺T、IL-2⁺CD8⁺T细胞阳性的占比为12/12、10/12、5/12。结论:AIM法在评价CD4⁺T细胞功能方面更为敏感,可作为ICS法的补充,两种方法联合使用可更全面地评估抗原特异性T淋巴细胞反应。     

CD8+ T-cell response against hepatitis C virus

CD8+ T-cell response is thought to be important for the control of hepatitis C virus (HCV) as well as for the liver cell injury caused by HCV infection. Studies on antigen-specific CD8+ T cells had long been hampered by lack of suitable techniques. Recently developed single-cell based assays, including peptide major histocompatibility complex (MHC) tetramer staining and intracellular cytokine staining, have greatly enhanced the opportunities for directly studying HCV-specific CD8+ T cells. Thanks to these novel assays the quantitative and qualitative nature of HCV-specific CD8+ T cells, including their number, phenotype, and effector functions, are starting to be revealed. However, much important information remains missing, including the signals for differentiation and migration of HCV-specific CD8+ T cells and the precise functions of antigen-specific effector cells in the virus-infected liver. The urgent need for effective immunotherapy and vaccines can not be met without a better understanding of the CD8+ T-cell response in HCV infection, which calls for a comprehensive strategy to study such cells directly using sensitive and quantitative assays.     

Haemorrhage-induced alterations in function and cytokine production of T cells and T cell subpopulations.

Haemorrhage produces alterations in macrophage, T and B cell function. In order to better define the mechanism for the effects of blood loss on immune response, we examined function of and cytokine production by purified T cells, CD4+ and CD8+ subpopulations after blood loss. Whereas T and CD4+ cells from control, unhaemorrhaged animals produced no alteration in proliferation when added to cultures of mitogen-stimulated splenocytes from normal mice, proliferation was decreased when T or CD4+ cells from haemorrhaged mice were included. The addition of CD8+ cells from haemorrhaged animals to mitogen-stimulated cultures reduced proliferation by approximately 50% more than that found when CD8+ cells from control, unhaemorrhaged animals were included. Supernatants of mitogen-stimulated splenocytes from haemorrhaged mice contained significantly less IL-2 and interferon-gamma (IFN-gamma) than did those from control, unhaemorrhaged mice. CD4+ populations from haemorrhaged mice produced significantly more IL-10, and significantly less IFN-gamma, than did CD4+ cells from control, unhaemorrhaged mice. There were no significant differences in IL-2, IL-4, IL-10 or IFN-gamma production by CD8+ cells from haemorrhaged or control mice. The present experiments demonstrate that haemorrhage affects both CD4+ and CD8+ T cell subsets. In particular, haemorrhage appeared to activate CD4+, Th2 cells, with concomitant suppression of the Th1 subpopulation. These results provide a mechanism which may contribute to the alterations in cytokine production previously described to occur following blood loss.     

A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-gamma HIV-specific immune responses

The single color IFN-γ ELISPOT assay has become a standard for assessing HIV-specific immune responses in HIV-infected subjects. However, recent data suggests that single cytokine detection for immune monitoring of HIV-infected individuals may not be sufficient to fully describe virus-specific immune responses. Here, we have designed and validated a dual color ELISPOT assay capable of detecting both IL-2 and IFN-γ secreting cells simultaneously in response to HIV antigens. We found that a cell input number of 200,000 cells/well provided a good balance between limited availability of cells due to blood volume restrictions and ability to detect all cytokine secretion patterns. The simultaneous detection of IL-2 and IFN-γ resulted in a decreased magnitude of IFN-γ but not IL-2 responses. Measures of intra- and inter-assay variability for the dual color ELISPOT assay were comparable to that seen for single cytokine ELISPOT assay with coefficients of variation below 20% for IL-2, IFN-γ and dual secretion. Although CD8+ T cells mediated most HIV-specific responses in infected subjects, CD4+ T cells mediated responses to HIV were also detected. Features of this assay such as high throughput, cell number requirement and cytokine choice should make this assay a valuable tool for screening for HIV-specific immune responses in several clinically relevant settings.     

Simultaneous assessment of antigen-stimulated cytokine production and memory subset composition of memory CD8 T cells

The functional identification of antigen-specific CD8 T cell populations is critical to understanding host responses to infection by intracellular pathogens. Furthermore, assessing the properties of protective memory CD8 T cell populations generated by immunization is necessary in the rational design of vaccines. Recently, a classification scheme was proposed in which memory CD8 T cells were divided into one of two distinct subsets, based on CD62L expression, that have different functional properties and protective capacities. Intracellular cytokine staining functionally identifies antigen-specific CD8 T cell populations after short in vitro stimulation with cognate peptide. This short stimulation, however, results in the cleavage of CD62L from the cell surface of antigen-specific CD8 T cells and precludes distinguishing CD62L(hi)- and CD62L(lo)-expressing memory cell subsets within this population. Here, we describe a method of preserving CD62L expression by the antigen-specific CD8 T cell population during coculture with antigen. This methodology allows for the identification and functional assessment of antigen-specific memory CD8 T cell populations, while simultaneously characterizing the memory subset composition of that population. Using this method, we directly identify differences in IL-2 production capacity by CD62L(hi)- and CD62L(lo)-expressing antigen-specific memory CD8 T cell populations.     

HIV/AIDS患者疾病进程与CD4+CD25hi调节性T细胞之间相关性研究

目的 通过分析不同阶段HIV感染者外周血CD4+CD25hi调节性T细胞(CD4+CD25hiregulatory T cells,Treg cells)与外周血免疫状态和病毒载量的相关性,探讨Treg细胞对HIV/AIDS发病进程的影响.方法 采集116例HIV感染者和21例正常人对照外周血,用4色流式细胞术进行CD4+和CD8+T细胞绝对数计数;用3色流式细胞术进行Treg细胞测定;用荧光定量PCR法进行HIVRNA载晕测定.实验数据用回归统计学方法和T检验方法进行分析.结果 HIV感染者外周血Treg细胞频率在HIV感染初期显著下降,之后随着疾病的进程逐渐升高.在CD4+T细胞大于300/μl的患者低于正常对照组,在CD4+T细胞小于100/μl的患者高于正常对照组,差异具有统计学意义.Treg细胞频率与CD4+T淋巴细胞绝对数和CD4+/CD8+之间均呈负相关.其相关系数r和P值各为r=-0.564,P＜0.001和r=-0.377,P＜0.001;Treg细胞频率与血浆HIV病毒载量呈正相关,其相关系数r=0.514.P＜0.001.结论 CD4+CD25hi Treg细胞可能是参与艾滋病免疫发病机理的重要细胞,在HIV感染发病进程的不同阶段具有不同的意义,其确切机制有待进行进一步研究.     

Immunisation with recombinant modified vaccinia virus Ankara expressing HIV-1 gag in HIV-1-infected subjects stimulates broad functional CD4+ T cell responses

Virus-specific CD4+ T cells with IL-2-secreting and/or proliferative capacity are detected readily in HIV-1-infected long-term nonprogressors and rarely in persons with untreated progressive infection. The contribution of these cells to viraemia control is uncertain, but this question might be addressed in clinical therapeutic vaccination studies. However, the quality of T helper responses induced by currently available HIV-1 vaccine candidates has not been explored in depth. We determined the effect of vaccination with modified vaccinia virus Ankara (MVA) expressing HIV-1 gag p24/p17 (MVA.HIVA) on HIV-1-specific CD4+ T cell responses in 16 chronically infected, highly active antiretroviral therapy (HAART)-treated subjects using CD8-depleted IFN-gamma ELISPOT assays, intracellular cytokine staining assays for IL-2 and IFN-gamma, and a CFSE-based proliferation assay. Gag-specific CD4+ T cell responses were significantly increased in magnitude and breadth after vaccination and targeted both known and new epitopes, several of which were also recognised by healthy HIV-uninfected volunteers immunised with the same vaccines. The frequencies of CD4+ T cells expressing IL-2 or IFN-gamma, alone or simultaneously, were also augmented. These findings indicate that functional virus-specific T helper cells can be boosted by vaccination in chronic HIV-1 infection. Further evaluation of their role in viraemia control is warranted.     

Flow cytometric detection of antigen-specific cytokine responses in lung T cells in a murine model of pulmonary inflammation.

Multiparameter flow cytometry was used to examine the cytokine responses of antigen-specific T lymphocytes isolated from the lungs of antigen-sensitized mice which developed pulmonary inflammation after aerosol challenge with ovalbumin (OA) (OA/OA). Lung T cells were stimulated in vitro with OA and anti-CD28 monoclonal antibody (mAb) in the presence of the secretion inhibitor, brefeldin A. T cell subsets were examined for intracellular cytokine expression using fluorochrome-labeled cell-surface specific and anti-cytokine antibodies. Antigen-specific responses resulted in significant numbers of CD4+ lung cells expressing cytoplasmic interleukin (IL)-2 (6%), IL-4 (1.5%), IL-5 (4%), and tumor necrosis factor (TNF)-alpha (11%), but not interferon (IFN)-gamma. Dual cytokine analyses demonstrated antigen-specific responses resulted in CD4+ T cells being positive for IL-2 and IL-4 or IL-2 and IL-5. TNF-alpha was the only antigen-specific cytokine response detected in CD8+ lung T cells after in vitro activation with OA. Cytokines in the supernatants of cultures activated with OA and anti-CD28 were measured by ELISA and the results confirmed the antigen-specific responses measured by flow cytometry. Polyclonal activation of lung T cells from OA/OA mice with 12-myristate 13-acetate (PMA), ionomycin, anti-CD3 mAb, and anti-CD28 mAb resulted in higher percentages of IL-2+ (43%) and IL-5+ (7%) CD4 cells when compared to CD4+ T cells from non-OA sensitized, challenged mice. CD8+ cells from OA/OA mice demonstrated intracellular staining for IL-2 (26%), TNF-alpha (55%), and IFN-gamma (37%), but not IL-4 or IL-5, after polyclonal activation. There is less agreement between intracellular cytokine staining of CD4+ T cells and cytokines released into the culture medium after polyclonal activation. Dual cytokine analyses of polyclonal-activated CD4+ cells demonstrated co-expression of IFN-gamma with IL-2, IL-4, or IL-5. T cells co-expressing IL-2 with IL-4 or IL-5 were also detected. These results demonstrate the utility of multiparameter flow cytometry to directly measure antigen-specific cytokine responses in subsets of T lymphocytes isolated from inflammatory sites.     

Double- and monofunctional CD4⁺ and CD8⁺ T-cell responses to Mycobacterium tuberculosis DosR antigens and peptides in long-term latently infected individuals

More than 2 billion individuals are latently infected with Mycobacterium tuberculosis (Mtb). Knowledge of the key Mtb antigens and responding T-cell subsets mediating protection against Mtb is critical for developing improved tuberculosis (TB) vaccines. We previously reported that Mtb DosR-regulon-encoded antigens are recognized well by human T cells in association with control of Mtb infection. The characteristics of the responding T-cell subsets, however, remained unidentified. We have therefore studied the cytokine production and memory phenotypes of Mtb DosR-regulon-encoded antigen-specific T cells from individuals who had been infected with Mtb decades ago, yet never developed TB (long-term latent Mtb-infected individuals). Using multi-parameter flow cytometry and intracellular cytokine staining for IFN-γ, TNF-α and IL-2, we found double and single cytokine-producing CD4(+) as well as CD8(+) T cells to be the most prominent subsets, particularly IFN-γ(+) TNF-α(+) CD8(+) T cells. The majority of these T cells comprised effector memory and effector T cells. Furthermore, CFSE labeling revealed strong CD4(+) and CD8(+) T-cell proliferative responses induced by several "immunodominant" Mtb DosR antigens and their specific peptide epitopes. These findings demonstrate the prominent presence of double- and monofunctional CD4(+) and CD8(+) T-cell responses in naturally protected individuals and support the possibility of designing Mtb DosR antigen-based TB vaccines.     

Interleukin-7 (IL-7)-induced proliferation of CD8+ T-chronic lymphocytic leukemia cells

Interleukin-7 (IL-7) is a growth factor for pro-B cells, pre-B cells, and thymocytes and is known to induce the proliferation of normal human peripheral T cells. Moreover, human B and T acute leukemia cells with immature surface markers proliferate in response to IL-7. Here we describe a case of T-chronic lymphocytic leukemia, in which the leukemic cells showed a proliferative response to human recombinant IL-7in vitro. The patient was a 74-year-old woman with anemia and thrombocytopenia, whose bone marrow was fibrosed and infiltrated with pathologic cells. Surface markers of the leukemic cells were CD2(+), CD3(+), CD5(+), CD7(+), CD8(+), and CD4(–). Both T-cell receptor -chain and -chain genes were found to be rearranged by immunogenotypic analysis. The leukemic cells proliferated in response to IL-7 dose dependently. The DNA synthesis of CLL cells was stimulated by not only IL-7 but also IL-2 and IL-4. The IL-7-induced proliferation was not inhibited by antibodies to IL-2 receptors or the anti-IL-4 antibody. These findings indicate that IL-7 may induce the proliferation of peripheral CD8⁺ T cells, even on its pathological counterpart.     

IL-15 ex vivo overcomes CD4+ T cell deficiency for the induction of human antigen-specific CD8+ T cell responses

CD4(+) Th cells are important for the induction and maintenance of antigen-specific CD8(+) T cell function, so their loss or dysfunction in HIV-infected or cancer patients could reduce the patients' ability to control viral infection. Previous work in murine systems indicated that IL-15 codelivered with vaccines could overcome CD4(+) Th cell deficiency for induction of functionally efficient CD8(+) T cells and maintenance of viral-specific CTLs, but its efficacy in helping primary human CD8(+) T cell responses is unknown. In the present study, a peptide-pulsed, DC-based human coculture ex vivo system was used to study the role of IL-15 in overcoming CD4(+) Th deficiency to elicit CD8(+) T cell responses in CD4-depleted PBMCs from healthy individuals and PBMCs from HIV-1-infected patients. We found that IL-15 could overcome CD4(+) Th deficiency to induce primary and recall memory CD8(+) T cell responses in healthy individuals. Moreover, in CD4-deficient, HIV-1-infected patients with diminished CD8(+) T cell responses, IL-15 greatly enhanced CD8(+) T cell responses to alloantigen. These results suggest that IL-15 may be useful in the development of therapeutic and preventive vaccines against cancers and viral infections in patients defective in CD4(+) Th cell.     

Cytokine production by cord blood mononuclear cells stimulated with cows milk proteins in vitro: interleukin-4 and transforming growth factor β-secreting cells detected in the CD45RO T cell population in children of atopic mothers

BACKGROUND: Food antigens from the maternal circulation may sensitize fetal T cells in utero and be an important determinant in the development of food allergy. METHODS: Here we have examined the spontaneous and recall response to cow's milk proteins of cord blood mononuclear cells (CBMC) of newborn children, using single cell ELISPOT assays. RESULTS: In term newborns, confirming previous studies, the spontaneous cytokine response of CBMC is dominated by IL-4, IL-5, IL-10, and as shown here for the first time, TGF-beta. For TGF-beta only, the response of samples from infants of atopic mothers was significantly lower than samples from infants of non-atopic mothers. In vitro stimulation of CBMC with bovine serum albumin, casein and beta-lactoglobulin resulted in a significant increase of all cytokine-secreting cells, again dominated by T helper type 2 (Th2) cytokines. There was a clear tendency for samples from infants of atopic mothers to have lower Th2 responses than samples from infants of non-atopic mothers, which was particularly significant for both IL-4 and TGF-beta. Spontaneous cytokine secreting cells were virtually absent in cord blood from infants < 34 weeks gestation, as were cows milk protein-induced responses, although they were readily detectable in samples from infants aged > 34 weeks. To explore whether the cytokine secreting cells were in the naive CD4+ CD45RA population or memory CD4+ CD45RO T cells, these subsets were purified by positive and negative selection and tested for spontaneous and cows milk protein-induced cytokine responses. Strikingly, although the responses were small, the CD45RO+ cells from children of atopic mothers showed significant spontaneous and antigen-specific IL-4 and TGF-beta responses, whereas the same population from infants of non-atopic mothers showed virtually no response. In addition CD45RA+ cells from infants of mothers with maternal atopy showed decreased IL-4 and TGF-beta responses, especially the latter. CONCLUSIONS: The cows milk antigen-specific IL-4 and TGF-beta responses preferentially seen in the memory cell subset of infants with a maternal history of atopy strongly suggests Th2 skewing to dietary antigens in utero.     

Rapid qualitative and quantitative analysis of T-cell responses in HIV-1-infected individuals receiving successful HAART and HIV-1 sero-negative controls: concomitant assessment of perforin, IFN-gamma and IL-4 secretion

The enzyme-linked immunospot (ELIspot) assay is a highly sensitive and valuable tool for determining the frequency of cytokine-secreting T cells. It is essential to determine both frequencies and functional capabilities of antigen-specific T cells, including cytokine secretion, degranulation, and cytotoxicity in order to obtain a fuller picture of the immune status of an individual. We describe here for the first time a perforin-release ELIspot assay which, when used in combination with IFN-gamma and IL-4 ELIspots, permits rapid assessment of these functional parameters for antigen-specific T cells. Whole antigen or peptides from HIV-1, recall and other viral antigens were used for in vitro stimulation. Anti-HIV-1 responses in treated chronically infected individuals were weak, both in terms of perforin and IFN-gamma production. Tetanus toxoid stimulation was associated with moderate perforin release and a predominantly type-2 IL-4 producing response, whilst herpes simplex virus antigen stimulation resulted in perforin release but only a weak type-1 IFN-gamma response. Anti-cytomegalovirus responses generated high levels of perforin in conjunction with IFN-gamma. Cytokines IL-2 and IL-12/IL-15 induced perforin release coupled with an IFN-gamma type-1 response. Perforin release strongly correlated with IFN-gamma production to individual influenza, Epstein-Barr virus or cytomegalovirus MHC class I restricted peptides, in an HIV-1 sero-negative cohort, indicating a cytolytic type-1 CD8+ T-cell response. Evaluation of immunogenicity and putative efficacy of candidate vaccines using IFN-gamma will not be as informative alone as when combined with perforin and IL-4 evaluations, which allow assessment of specific cytotoxic potential without extensive cell culture.     

Reversible CD8 expression induced by common cytokine receptor gamma chain-dependent cytokines in a cloned CD4(+) T(h)1 cell line

T cells that are intrathymically lineage committed are believed to maintain their CD4 or CD8 co-receptor expression. Here, we investigated whether intrathymic lineage commitment involves irreversible genetic modification or whether co-receptor expression can be reprogrammed depending on external stimuli. The CD4(+) T(h)1 clone 2D6 established from splenic T cells as an IL-12-dependent line survived in culture with IL-2, IL-7 or IL-15 alone. Surprisingly, CD8 expression occurred in 2D6 cells upon replacement of IL-12 with any one of the three cytokines that stimulate the common cytokine receptor gamma chain, yielding CD4(+)CD8(+) 2D6 cells. CD8 expression declined when IL-2 was replaced with IL-12 and CD8 induction was inhibited when IL-12 was included in IL-2 or IL-7 culture. Our observations show that even a lineage-committed mature T cell can be reprogrammed for co-receptor expression in response to particular external stimuli.