Study design may explain discrepancies in GB virus C effects on interferon-γ and interleukin-2 production and CD38 expression in T lymphocytes

Bhattarai, Nirjal; Stapleton, Jack T

doi:10.1590/S0074-02762012000400023

LETTER TO THE EDITOR

Study design may explain discrepancies in GB virus C effects on interferon-γ and interleukin-2 production and CD38 expression in T lymphocytes

Nirjal Bhattarai^{I, II}; Jack T StapletonI, II, ⁺ + Corresponding author: jack-stapleton@uiowa.edu

^IIowa City Department of Veterans Affairs, Iowa, IA, USA

^IIUniversity of Iowa, Iowa, IA, USA

Recently, Baggio-Zappia et al. (2011) reported the effects of GB virus C (GBV-C) on interleukin (IL)-2 and interferon (IFN)-γ in CD4 and CD8 T cells. GBV-C coinfection was not associated with lower immune activation in human immunodeficiency virus (HIV)-positive individuals as measured by CD38 expression per cell and IL-2 and IFN-γ expressing cells in their study. We agree with Baggio-Zappia et al. (2011) that "several factors, such as differences in the study populations, the stage of HIV infection and the time of GBV-C acquisition, may account for the discrepant results reported by different research groups". However, two major factors may confound interpretation of the results reported by Baggio-Zappia et al. and provide the most likely explanation for the discrepant results observed between this study and previous reports (Maidana-Giret et al. 2009, Baggio-Zappia et al. 2011).

First, approximately 85% of the subjects studied were on potent combination antiretroviral therapy (cART) and had nondetectable HIV RNA levels in their plasma. cART lowers T cell activation, raises CD4 counts, decreases HIV VL and thus interferes significantly with other factors associated with HIV disease progression (Autran et al. 1997, Carcelain et al. 2001, Lederman 2001). Since the vast majority of subjects in the study of Baggio-Zappia et al. (2011) were effectively treated by cART, it is not surprising that GBV-C did not have an observed effect on the clinical variables studied (CD4, HIV VL etc.).

Secondly, Baggio-Zappia et al. (2011) froze the peripheral blood mononuclear cells (PBMCs) prior to conducting in vitro studies of activation, IL-2 and IFN-γ cytokine production. Freeze-thawing PBMCs can alter detection of both surface marker expression and T cell function (Kvarnstrom et al. 2004, Mallone et al. 2011). By comparison, several groups found a reduction in CD38 on CD4 and/or CD8 T cells in HIV-infected subjects using fresh PBMCs (Bhattarai et al. 2011, Maidana-Giret et al. 2009). In addition, HIV entry (chemokine) receptors CCR5 and CXCR4 are upregulated on CD4 and CD8 T cells following T cell activation (Smith et al. 2002). Three independent groups found reduced levels of CCR5 on CD4 cells using fresh, non-frozen PBMCs and one of these studies also found reduced levels of CXCR4 on CD4 cells (Nattermann et al. 2003, Maidana-Giret et al. 2009, Schwarze-Zander et al. 2010). Finally, IL-2 interactions with GBV-C have been demonstrated in vitro (George et al. 2003, Bhattarai 2011) and in vivo (Stapleton et al. 2009) using fresh, non-frozen cells for flow cytometry studies.

Since cART will lower detection of cell surface markers of T cell activation, making detection of a diminution by GBV-C more difficult to detect, and since frozen PBMCs can alter levels of T cell activation markers, we believe that the study of Baggio-Zappia et al. (2011) does not negate the prior studies that used fresh PBMCs to evaluate T cell activation. However, the results illustrate the need for more direct and thorough studies of GBV-C on T cell function in individuals with HIV-infection.

REPLY

Comments in regards to Bhattarai and Stapleton's letter

Recently, our group reported the findings of a study that evaluated the effect of GBV-C viremia on IL-2 and IFN-γ production in CD4, CD8 and γΔ T cells and CD38 expression in CD4 and CD8 T cells. Our results did not find an association of GBV-C viremia with lower immune activation in human immunodeficiency virus (HIV)-positive or GBV-C-hepatitis C virus (HCV) co-infected individuals (Baggio-Zappia et al. 2011). In agreement, other studies conducted with different cohorts of HIV-infected patients did not found a beneficial effect of GBV-C on the course of HIV disease (Brumme et al. 2002, Quiros-Roldan et al. 2002, Bjorkman et al. 2004). N Bhattarai and JT Stapleton point that the results found in the study are a result of the ART and to the use of frozen PBMCs to conduct the in vitro experiments.

We agree with N Bhattarai and JT Stapleton that ART lowers T cell activation, raises CD4 T cell counts, decreases HIV VL, and interferes significantly with other factors associated with HIV disease progression, as numerous studies previously demonstrated (Autran et al. 1997, Carcelain et al. 2001, Lederman et al. 2001), however, in this study, all the patients presented a considering degree of immune activation, as demonstrated by the evaluation of CD38 activation marker, independent of the use of ART. In addition, in order to address if the lack of statistical significance was the use of ART, we performed two analyses, considering HIV-treated and HIV-non treated patients and we found the same results (data not shown). Interestingly, Antonucci et al. evaluated the relationship between GBV-C infection and response to antiretroviral therapy in a cohort of 400 HIV-infected patients found that those patients who were GBV-C infected presented a lower risk of HIV rebound when compared with those who were GBV-C negative, showing that an effect of the virus was observed in spite of the ART therapy. In the same study, the authors found that the probability of achieving initial virological success or CD4 T cell count response after HAART did not differ between GBV-C negative and GBV-C positive subjects.

In our study (Baggio-Zappia et al. 2011) frozen (PBMCs was used to conduct the in vitro studies of activation, IL-2 and IFN-γ cytokine. As an experienced group in the methodology, we have already conducted experiments with fresh PBMCs and the same results were obtained. In addition to our experience, other studies demonstrated that cytokine release in vitro is not significantly different in fresh or cryopreserved PBMCs (Friberg et al. 1994, Venet et al. 2010). Another point to be considered regarding CD38 is that in the present study, we evaluated the quantitative expression of the immune activation marker, not only if the cells were expressing it, as performed in previous studies (Maidana-Giret et al. 2009), and this could explain the differences of the results, besides the differences between chronically and early infected patients.

Our study has never had the intention to negate the prior studies; instead it adds new data that corroborate the need to conduct more detailed studies that consider the stages of the HIV infection and also, the role of the co-infection with other viruses such as HCV in the context of the HIV disease.

REFERENCES

Giovana Lotici Baggio-Zappia

Aline de Jesus Barbosa

Milena Karina Coló Brunialti

Reinaldo Salomão

Disciplina de Infectologia,

Laboratório de Virologia e Imunologia,

Universidade Federal de São Paulo, São Paulo, SP, Brasil

Celso Francisco Hernandes Granato

Disciplina de Infectologia,

Laboratório de Virologia e Imunologia,

Universidade Federal de São Paulo, São Paulo, SP,

Fleury Medicina Diagnóstica, São Paulo, SP, Brasil

Received 4 November 2011

Accepted 11 January 2012

Autran B, Carcelain G, Li TS, Blanc C, Mathez D, Tubiana R 1997. Positive effects of combined antiretroviral therapy on CD4⁺ T cell homeostasis and function in advanced HIV disease. Science 277: 112-116.
Baggio-Zappia GL, Barbosa Ade J, Brunialti MKC, Salomão R, Granato CFH 2011. Influence of GB virus C on IFN-γ and IL-2 production and CD38 expression in T lymphocytes from chronically HIV-infected and HIV-HCV-co-infected patients. Mem Inst Oswaldo Cruz 106: 662-669.
Bhattarai N, Rydze RT, Xiang J, Landay A, McLinden J, Stapleton JT 2011. GBV-C infection effects on T cell activation and IL-2 secretion can be mediated by the envelope glycoprotein E2, 6th International AIDS Society Conference on HIV Pathogenesis, Treatment and Prevention, Rome, 150 pp.
Carcelain G, Debre P, Autran B 2001. Reconstitution of CD4⁺ T lymphocytes in HIV-infected individuals following antiretroviral therapy. Curr Opin Immunol 13: 483-488.
George SL, Xiang J, Stapleton JT 2003. Clinical isolates of GB virus type C vary in their ability to persist and replicate in peripheral blood mononuclear cell cultures. Virology 316: 191-201.
Kvarnstrom M, Jenmalm MC, Ekerfelt C 2004. Effect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles analysed with three common assays: an overall decrease of interleukin-4. Cryobiology 49: 157-168.
Lederman MM 2001. Immune restoration and CD4⁺ T-cell function with antiretroviral therapies. AIDS 15 (Suppl. 2): S11-S15.
Maidana-Giret MT, Silva TM, Sauer MM, Tomiyama H, Levi JE, Bassichetto KC 2009. GB virus type C infection modulates T-cell activation independently of HIV-1 viral load. AIDS 23: 2277-2287.
Mallone R, Mannering SI, Brooks-Worrell BM, Durinovic-Bello I, Cilio CM, Wong FS 2011. Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society. Clin Exp Immunol 163: 33-49.
Nattermann J, Nischalke HD, Kupfer B, Rockstroh J, Hess L, Sauerbruch T 2003. Regulation of CC chemokine receptor 5 in hepatitis G virus infection. AIDS 17: 1457-1462.
Schwarze-Zander C, Neibecker M, Othman S, Tural C, Clotet B, Blackard JT 2010. GB virus C coinfection in advanced HIV type-1 disease is associated with low CCR5 and CXCR4 surface expression on CD4(⁺) T-cells. Antiviral therapy 15: 745-752.
Smith KY, Kumar S, Pulvirenti JJ, Gianesin M, Kessler HA, Landay A 2002. CCR5 and CXCR4 expression after highly active antiretroviral therapy (HAART). J Acquir Immune Defic Syndr 30: 458-460.
Stapleton JT, Chaloner K, Zhang J, Klinzman D, Souza IE, Xiang J 2009. GBV-C viremia is associated with reduced CD4 expansion in HIV-infected people receiving HAART and interleukin-2 therapy. AIDS 23: 605-610.
Antonucci G, Girardi E, Cozzi-Lepri A, Capobianchi MR, Morsica G, Pizzaferri P, Ladisa N, Sighinolfi L, Chiodera A, Solmone M, Lalle E, Ippolito G, Monforte A, HepaICoNA Study Group, ICoNA Study Group 2005. Antivir Ther 10: 109-117.
Autran B, Carcelain G, Li TS, Blanc C, Mathez D, Tubiana R 1997. Positive effects of combined antiretroviral therapy on CD4⁺ T cell homeostasis and function in advanced HIV disease. Science 277: 112-116.
Baggio-Zappia GL, Barbosa Ade J, Brunialti MKC, Salomão R, Granato CFH 2011. Influence of GB virus C on IFN-γ and IL-2 production and CD38 expression in T lymphocytes from chronically HIV-infected and HIV-HCV-co-infected patients. Mem Inst Oswaldo Cruz 106: 662-669.
Björkman P, Flamholc L, Nauclér A, Molnegren V, Wallmark E, Widell A 2004. GB virus C during the natural course of HIV-1 infection: viremia at diagnosis does not predict mortality. AIDS 18: 877-886.
Brumme ZL, KJ Chan, Dong WW, Mo T, Wynhoven B, Hogg RS, Montaner JS, O'Shaughnessy MV, Harrigan 2002. No association between GB virus-C viremia and virological or immunological failure after starting initial antiretroviral therapy. AIDS 16: 1929-1933.
Carcelain G, Debre P, Autran B 2001. Reconstitution of CD4⁺ T lymphocytes in HIV-infected individuals following antiretroviral therapy. Curr Opin Immunol 13: 483-488.
Friberg D, Bryant J, Shannon W, Whiteside TL 1994. In vitro cytokine production by normal human peripheral blood mononuclear cells as a measure of immunocompetence or the state of activation. Clin Diag Lab Immun 1: 261-268.
Lederman MM 2001. Immune restoration and CD4⁺ T-cell function with antiretroviral therapies. AIDS 15 (Suppl. 2): S11-S15.
Maidana-Giret MT, Silva TM, Sauer MM, Tomiyama H, Levi JE, Bassichetto KC 2009. GB virus type C infection modulates T-cell activation independently of HIV-1 viral load. AIDS 23: 2277-2287.
Quiros-Roldan E, Maroto MC, Torti C, Moretti F, Casari S, Pan A, Carosi G 2002. No evidence of benefical effect of GB virus type C infection on the course of HIV infection. AIDS 16: 1430-1431.
Venet F, Malcus C, Ferry T, Politevin F, Monneret G, 2010. Percentage of regulatory T cells CD4+CD25+CD127 in HIV-infected patients is not reduced after cryopreservation. J Immun Meth 357: 55-58.

+

Corresponding author:

jack-stapleton@uiowa.edu

Publication Dates

Publication in this collection
01 June 2012
Date of issue
June 2012

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] Autran B, Carcelain G, Li TS, Blanc C, Mathez D, Tubiana R 1997. Positive effects of combined antiretroviral therapy on CD4⁺ T cell homeostasis and function in advanced HIV disease. Science 277: 112-116.

[2] Baggio-Zappia GL, Barbosa Ade J, Brunialti MKC, Salomão R, Granato CFH 2011. Influence of GB virus C on IFN-γ and IL-2 production and CD38 expression in T lymphocytes from chronically HIV-infected and HIV-HCV-co-infected patients. Mem Inst Oswaldo Cruz 106: 662-669.

[3] Bhattarai N, Rydze RT, Xiang J, Landay A, McLinden J, Stapleton JT 2011. GBV-C infection effects on T cell activation and IL-2 secretion can be mediated by the envelope glycoprotein E2, 6th International AIDS Society Conference on HIV Pathogenesis, Treatment and Prevention, Rome, 150 pp.

[4] Carcelain G, Debre P, Autran B 2001. Reconstitution of CD4⁺ T lymphocytes in HIV-infected individuals following antiretroviral therapy. Curr Opin Immunol 13: 483-488.

[5] George SL, Xiang J, Stapleton JT 2003. Clinical isolates of GB virus type C vary in their ability to persist and replicate in peripheral blood mononuclear cell cultures. Virology 316: 191-201.

[6] Kvarnstrom M, Jenmalm MC, Ekerfelt C 2004. Effect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles analysed with three common assays: an overall decrease of interleukin-4. Cryobiology 49: 157-168.

[7] Lederman MM 2001. Immune restoration and CD4⁺ T-cell function with antiretroviral therapies. AIDS 15 (Suppl. 2): S11-S15.

[8] Maidana-Giret MT, Silva TM, Sauer MM, Tomiyama H, Levi JE, Bassichetto KC 2009. GB virus type C infection modulates T-cell activation independently of HIV-1 viral load. AIDS 23: 2277-2287.

[9] Mallone R, Mannering SI, Brooks-Worrell BM, Durinovic-Bello I, Cilio CM, Wong FS 2011. Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society. Clin Exp Immunol 163: 33-49.

[10] Nattermann J, Nischalke HD, Kupfer B, Rockstroh J, Hess L, Sauerbruch T 2003. Regulation of CC chemokine receptor 5 in hepatitis G virus infection. AIDS 17: 1457-1462.

[11] Schwarze-Zander C, Neibecker M, Othman S, Tural C, Clotet B, Blackard JT 2010. GB virus C coinfection in advanced HIV type-1 disease is associated with low CCR5 and CXCR4 surface expression on CD4(⁺) T-cells. Antiviral therapy 15: 745-752.

[12] Smith KY, Kumar S, Pulvirenti JJ, Gianesin M, Kessler HA, Landay A 2002. CCR5 and CXCR4 expression after highly active antiretroviral therapy (HAART). J Acquir Immune Defic Syndr 30: 458-460.

[13] Stapleton JT, Chaloner K, Zhang J, Klinzman D, Souza IE, Xiang J 2009. GBV-C viremia is associated with reduced CD4 expansion in HIV-infected people receiving HAART and interleukin-2 therapy. AIDS 23: 605-610.

[14] Antonucci G, Girardi E, Cozzi-Lepri A, Capobianchi MR, Morsica G, Pizzaferri P, Ladisa N, Sighinolfi L, Chiodera A, Solmone M, Lalle E, Ippolito G, Monforte A, HepaICoNA Study Group, ICoNA Study Group 2005. Antivir Ther 10: 109-117.

[15] Autran B, Carcelain G, Li TS, Blanc C, Mathez D, Tubiana R 1997. Positive effects of combined antiretroviral therapy on CD4⁺ T cell homeostasis and function in advanced HIV disease. Science 277: 112-116.

[16] Baggio-Zappia GL, Barbosa Ade J, Brunialti MKC, Salomão R, Granato CFH 2011. Influence of GB virus C on IFN-γ and IL-2 production and CD38 expression in T lymphocytes from chronically HIV-infected and HIV-HCV-co-infected patients. Mem Inst Oswaldo Cruz 106: 662-669.

[17] Björkman P, Flamholc L, Nauclér A, Molnegren V, Wallmark E, Widell A 2004. GB virus C during the natural course of HIV-1 infection: viremia at diagnosis does not predict mortality. AIDS 18: 877-886.

[18] Brumme ZL, KJ Chan, Dong WW, Mo T, Wynhoven B, Hogg RS, Montaner JS, O'Shaughnessy MV, Harrigan 2002. No association between GB virus-C viremia and virological or immunological failure after starting initial antiretroviral therapy. AIDS 16: 1929-1933.

[19] Carcelain G, Debre P, Autran B 2001. Reconstitution of CD4⁺ T lymphocytes in HIV-infected individuals following antiretroviral therapy. Curr Opin Immunol 13: 483-488.

[20] Friberg D, Bryant J, Shannon W, Whiteside TL 1994. In vitro cytokine production by normal human peripheral blood mononuclear cells as a measure of immunocompetence or the state of activation. Clin Diag Lab Immun 1: 261-268.

[21] Lederman MM 2001. Immune restoration and CD4⁺ T-cell function with antiretroviral therapies. AIDS 15 (Suppl. 2): S11-S15.

[22] Maidana-Giret MT, Silva TM, Sauer MM, Tomiyama H, Levi JE, Bassichetto KC 2009. GB virus type C infection modulates T-cell activation independently of HIV-1 viral load. AIDS 23: 2277-2287.

[23] Quiros-Roldan E, Maroto MC, Torti C, Moretti F, Casari S, Pan A, Carosi G 2002. No evidence of benefical effect of GB virus type C infection on the course of HIV infection. AIDS 16: 1430-1431.

[24] Venet F, Malcus C, Ferry T, Politevin F, Monneret G, 2010. Percentage of regulatory T cells CD4+CD25+CD127 in HIV-infected patients is not reduced after cryopreservation. J Immun Meth 357: 55-58.