Microbial Diversity

eji.201545940.pdf

Home >Biology homework help >Microbial Diversity

Eur. J. Immunol. 2015. 45: 3107–3113 Immunity to infectionDOI: 10.1002/eji.201545940 3107

SHORT COMMUNICATION

Gut commensal microbes do not represent a dominant antigenic source for continuous CD4+ T-cell activation during HIV-1 infection

Kathrin Zimmermann1, Sonia Bastidas1, Leandra Knecht1, Herbert Kuster2, Stephan R. Vavricka4, Huldrych F. Günthard2,3

and Annette Oxenius1

1 Institute of Microbiology, ETH Zurich, Zurich, Switzerland 2 Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich,

University of Zurich, Zurich, Switzerland 3 Institute of Medical Virology, University of Zurich, Zurich, Switzerland 4 Division of Gastroenterology and Hepatology, Triemli Hospital, Zurich, Switzerland

Chronic immune activation is a hallmark of HIV-1 infection; specifically, the activation of T cells has predictive value for progression to AIDS. The majority of hyperactivated T cells are not HIV-specific and their antigenic specificities remain poorly understood. Translocation of gut luminal microbial products to systemic sites contributes to chronic immune activation during HIV-1 infection, but how it affects (TCR-dependent) immune activation remains elusive. We hypothesized that gut luminal antigens foster activation of CD4+ T cells with specificities for commensal bacterial antigens, thereby contribut- ing to the pool of activated CD4+ T cells in the circulation of HIV-1 infected individu- als. To test this hypothesis, we quantified the frequencies of gut microbe-specific CD4+

T cells by cytokine production upon restimulation with selected gut commensal microbial antigens. Contrary to our hypothesis, we did not observe increased but rather decreased frequencies of gut microbe-specific CD4+ T cells in HIV-1 infected individuals compared to healthy controls. We conclude that the increased activation status of circulating CD4+

T cells in HIV-1 infected individuals is not driven by CD4+ T cells with specificities for commensal bacterial antigens.

Keywords: Gut microbe-specific CD4+ T-cell responses · IBD and HIV-1 associated enteropathy · Microbial translocation · Systemic immune activation

�Additional supporting information may be found in the online version of this article at thepublisher’s web-site

Introduction

Chronic activation of the immune system is a major determinant of progression to AIDS [1] and particularly the level of hyperac- tivated T cells is considered as best prognostic marker for disease

Correspondence: Dr. Annette Oxenius e-mail: [email protected]

progression [2]. HIV-1 infection and replication is the main driv- ing force of persistent, pathological level of T-cell activation [1, 3] but HIV-1 specific T cells account for only a small fraction of acti- vated T cells [4–6], implicating substantial “bystander” activation. Multiple mechanisms are considered to contribute to the genesis of such extensive immune activation, many of them are related to HIV-1 associated immunodeficiency [7, 8], sustained inflamma- tion [1], and microbial translocation [9].

3108 Kathrin Zimmermann et al. Eur. J. Immunol. 2015. 45: 3107–3113

HIV-1-related immunodeficiency leads to the reactivation of ubiquitous symbiotic viruses (e.g. herpes viruses), generating HIV- unrelated antigens [7, 8]. Furthermore, the rapid and irreversible depletion of intestinal CD4+ T cells, in particular Th17 cells that are essential for maintaining barrier function [10, 11] during HIV-1 infection impair the integrity of the intestinal barrier and enable the translocation of commensal microbes (or their prod- ucts) from the gut lumen to systemic sites [9]. Thus, HIV-1 repli- cation, HIV-1-associated immunodeficiency [7, 8], and microbial translocation [9] fuel a milieu rich of viral and bacterial pathogen- associated molecular patterns and proteins that have the capacity to activate innate and adaptive immune cells by engaging pat- tern recognition receptors and rearranged high affinity antigen receptors, respectively. Indeed, constant triggering of these recep- tors in combination with the ensuing cytokine secretion likely all contribute to HIV-1 associated hyper-activation of the immune system, which is best described for T cells. Interestingly, causes for activation of non-HIV-specific CD4+ and CD8+ T cells seem to differ with respect to involvement of TCR engagement: whereas CD8+ T cells can easily be activated by cytokines (e.g. IL-15) in a TCR-independent manner [12], the activation of CD4+ T cells seems to depend more on TCR-dependent cognate antigen sens- ing [13, 14]. DCs, among other cells, adopt a more mature and activated phenotype [13, 15, 16], promoting TCR-mediated CD4+

T-cell activation, in particular in situations of low antigen abun- dance. Nevertheless, the antigenic specificities of non HIV-specific hyperactivated CD4+ T cells remain poorly defined. We have pre- viously demonstrated that HIV-1 replication leads to the activa- tion of herpes virus-specific CD4+ T cells even in the absence of detectable herpes virus replication [13, 17]. However, herpes virus-specificity only accounts for a small fraction of non-HIV- specific activated CD4+ T cells during HIV-1 infection. Here, we hypothesized that activated CD4+ T cells might also compromise specificities for gut microbial antigens, based on the increased exposure of systemic immune cells to gut commensal microbes [9]. To test this hypothesis, we measured the frequencies of circulat- ing gut-microbe specific CD4+ T cells with specificity for selected gut commensal microbial antigens. We included not only HIV- 1 patients but also inflammatory bowel disease (IBD) patients to compare to another condition of sustained gastrointestinal inflam- mation [18, 19].

Results and discussion

CD4+ T cells specific for gut commensal microbes are present in blood of healthy individuals

The structural and immunological integrity of the intestinal bar- rier is crucial for the confinement of commensal microbes to the gut lumen and for a strict compartmentalization between the gas- trointestinal and systemic immune system—at least in clean exper- imental conditions such as specific-pathogen free mice [20]. In contrast, humans are repeatedly experiencing episodes of compro- mised gastrointestinal barrier integrity, provoked by, e.g. gastroin-

testinal tract (GIT) infections and behavioral patterns affecting the integrity of the GIT barrier function [21]. These episodes are asso- ciated with the exposure of gut microbial products to immune cells at systemic sites, documented by serum IgG antibodies with speci- ficities for gut microbial antigens being present in healthy donors [22, 23]. It is unclear whether this also holds true for CD4+ T cells with gut microbial specificities. Hence we investigated this ques- tion by analyzing cytokine production upon restimulation with lysates from gut commensal microbes.

Indeed, CD4+ T cells isolated from blood produced IFN-γ and IL-17 upon restimulation with a mixture of Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Enterococcus faecalis (E. faecalis), and Bacteroides thetaiotaomicron (B. thetaiotaomi- cron) derived bacterial lysates in healthy individuals, albeit at very low frequencies (Fig. 1A and B). As only very few of the bacte- rial lysate-responsive IFN-γ producing cells were CD4 negative and expressed CD161 (identifying invariant Mucosal-associated Invari- ant T cells), we conclude that CD4+ T cells are the predominant source of these cytokines (Fig. 1C). Furthermore, the responses were dependent on TCR-MHC interaction. MHC II blocking dur- ing restimulation with E. coli lysate led to significant reduction of IFN-γ production (Fig. 1D, left). Stimulating CD4+ T cells with bacterial lysates could include responses specific for conserved intracellular bacterial proteins that are shared between many bac- terial species and therefore not being an identifier for gut com- mensals. Since CD4+ T cells produced much less IFN-γ upon res- timulation with a lysate derived from the non-human-related soil bacterium B. japonicum compared to E. coli lysate, we conclude that gut microbe-specific CD4+ T cells are indeed found in the cir- culation of healthy humans and that our experimental approach is able to detect those responses (Fig. 1D, right). Restimulation of CD4+ T cells with lysates derived from E. coli, K. pneumo- niae, and C. albicans revealed relatively high frequencies of IFN-γ, IL-17, and IL-2 producing CD4+ T cells (Fig. 1E–G), consistent with previous reports documenting systemic IgG responses against gut commensal microbes in the serum of healthy individuals [22, 23]. Together, these data imply that the strict compartmen- talization between gut mucosal and systemic immune responses toward commensal microbes is—at least to some extent—broken in healthy human individuals most likely in response to tran- sient episodes of bacterial translocation [21]. Because CD4+ T-cell responses specific for E. faecalis and B. thetaiotaomicron were gen- erally close to the detection limit, we did not include these lysates in the further experiments.

Gut microbe-specific CD4+ T cells do not contribute to activated T cells during HIV-1 infection

The intestinal barrier integrity is severely impaired in HIV-1 infected individuals, allowing translocation of gut commensal luminal products into the systemic circulation [9]. We therefore examined whether this is associated with higher frequencies of systemic CD4+ T cells with gut microbial specificities in HIV-1 patients (Supporting Information Table 1). To our surprise, the

Eur. J. Immunol. 2015. 45: 3107–3113 Immunity to infection 3109

Figure 1. Gut commensal microbe-specific CD4+ T cells are present in the blood of healthy individuals. Peripheral blood CD4+ T cells from healthy individuals were stimulated overnight with a mixture or individual lysates from E. coli, K. pneumoniae, E. faecalis, B. thetaiotaomicron, C. albicans or the plant symbiont B. Japonicum in the presence of autologous CD14+ monocytes as indicated in the figure. (A) Representative FACS dot plot show intracellular IFN-γ and IL-17 production of CD4+ T cells. Target CD4+ T cells were sequentially gated on lymphocytes, singlets, live CD3+ CD4+

cells and a minimum of 300 000 events were acquired for each sample. (B) Bar graph represents the frequency (‰) of IFN-γ+ (left) and IL-17+ (right) CD4+ T cells (N = 15). Dots represent individual values and bars represent the median value, statistical significance was evaluated by the unpaired t-test. Data shown are representative of four independent experiments. (C) Representative FACS dot plot showing intracellular IFN-γ production in CD3+ cells (top) that had been gated on live lymphocytes after doublet exclusion and plotting of CD4 versus CD161 on IFN-γ+ cells (bottom). (D) Bar graph depicts IFN-γ-spot forming units (SFU) per 106 CD4+ T cells upon stimulation with E. coli lysate in the presence or absence of MHC II blocking antibody (left, N = 6) as well as E. coli and B. Japonicum lysates (right, N = 12). Dots represent individual values and bars represent the median value, statistical significance was evaluated by the paired t-test, ***p < 0.001, *p < 0.05. Data shown are representative of two (left) and four (right) independent experiments. (E–G) Bar graphs depict IFN-γ (E), IL-17 (F), and IL-2 (G) SFU per 106 CD4+ T cells upon stimulation with E. coli, K. pneumoniae, E. faecalis, B. thetaiotaomicron, and C. albicans lysates (N = 16). Data shown are representative of >20 independent experiments. Statistical significance was evaluated by the two-tailed paired t-test; ***p < 0.001, **p < 0.01.

frequencies of INF-γ producing CD4+ T cells specific for E. coli, K. pneumoniae, and C. albicans were significantly lower in HIV- 1 patients compared to healthy controls (Fig. 2A) and a similar trend toward lower frequencies of E. coli-, K. pneumoniae-, and C. albicans-specific CD4+ T cells producing IL-17 and IL-2 was observed (Fig. 2B and C).

These results indicate that gut microbe-specific CD4+ T cells— despite presumably prolonged availability of gut microbial anti- gens at systemic sites during HIV-1 infection—are not a major con- stituent of activated CD4+ T cells in the blood of HIV-1 patients. This is likely caused by several factors: during active HIV-1 repli- cation gut commensal-specific CD4+ T cells might be targets for infection which might curtail their abundance. We therefore delib- erately enrolled HIV-1 infected patients on successful antiretro- viral combination therapy (cART) for this analysis (Supporting Information Table 1), reflected by relative low levels of systemic immune activation as determined by coexpression of HLA-DR and CD38 on peripheral blood T cells (Supporting Information Fig. 1) and CD4 T-cell counts were restored to relative high lev- els (Supporting Information Table 1). Furthermore, CD4+ T-cell

responses specific for E. coli (Fig. 2D) were highly elevated in a patient exhibiting a GIT infection at the time of blood withdrawal, likely reflecting a transient decrease of the intestinal barrier func- tion along with expansion of microbiota-specific T cells [24]. This observation suggests that immune competence in HIV-1 infected individuals is not compromised to an extent that it would prevent priming of gut microbe-specific CD4+ T-cell responses. Another potential factor that might substantially curtail the frequencies of gut-microbe-specific CD4+ T cells at systemic sites in HIV-1 patients is the severe and pronounced depletion of intestinal CD4+

T cells [10, 11] whose recovery is not achieved with cART [25].

IBD patients exhibit comparable levels of gut commensal-specific CD4+ T cells as healthy controls

IBD patients suffer from sustained gastrointestinal tract inflam- mation that compromises intestinal barrier function and is asso- ciated with pronounced microbial translocation [18, 19]. Impor- tantly, this pathology is accompanied by local but not systemic

3110 Kathrin Zimmermann et al. Eur. J. Immunol. 2015. 45: 3107–3113

Figure 2. No elevated abundance of systemic CD4+ T cells with speci- ficities for gut commensal microbes in HIV-1 patients compared to healthy controls. CD4+ T cells from the blood of HIV-1 patients (N = 22) of which one experienced a GIT infection (D) at the time of blood withdrawal and of healthy donors (N = 16) were restimulated overnight with lysates derived from E. coli, K. pneumoniae, and C. albicans in the presence of autologous CD14+ monocytes and subjected to IFN-γ (A and D), IL-17 (B), and IL-2 (C) ELISpot assays to enumerate cytokine pro- ducing cells. Dots represent individual SFU per 106 CD4+ T cells after background substraction and bars represent the median value. Data shown are representative of >14 independent experiments. Statistical significance was evaluated by the nonparametric Wilcoxon rank sum test; ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 3. Comparable levels of systemic gut commensal-specific CD4+

T cells in IBD patients and healthy controls. CD4+ T cells from the blood of IBD patients (N = 19) and healthy donors (N = 14) were restimulated overnight with lysates derived from E. coli, K. pneumoniae, and C. albicans in the presence of autologous CD14+ monocytes and subjected to IFN-γ (A), IL-17 (B), and IL-2 (C) ELISpot assays to enumerate cytokine pro- ducing cells. Dots represent individual SFU per 106 CD4+ T cells after background substraction and bars represent the median value. Data shown are representative of 19 independent experiments. Statistical significance was evaluated by the nonparametric Wilcoxon rank sum test; ***p < 0.001, **p < 0.01, *p < 0.05.

immune activation and thereby represents an ideal condition to analyze whether the presence of gut luminal products in the cir- culation translates into high levels of systemic CD4+ T cells with corresponding specificities. Along that line, we and others have demonstrated that the augmented exposure of systemic immune cells to constituents of the gut commensal microbiota leads to enhanced levels of systemic microbe-specific antibody responses [22, 23]. Interestingly, peripheral CD4+ T cells from IBD patients were not substantially enriched for specificities toward gut com- mensal microbes (Fig. 3A–C), except the frequency of IL-2 pro- ducing CD4+ T cells specific for E. coli were slightly elevated in IBD patients compared to healthy controls.

Excessive GIT homing of CD4+ T cells is observed in IBD- associated pathology [26] and could explain why no increased

Eur. J. Immunol. 2015. 45: 3107–3113 Immunity to infection 3111

frequencies of gut microbe-specific CD4+ T cells are systemically detectable in IBD patients compared to healthy controls. Such excessive gut homing does probably not explain the absence of gut microbe-specific CD4+ T cells in the circulation of HIV-1 patients, since gut homing of CD4+ T cells is reported to be impaired in HIV-1 patients [27]. Furthermore, our own findings suggest that bacteria-specific IL-17 producing CD4+ T cells do not express high levels of gut homing receptors α4β7 (at least in healthy donors) (Supporting Information Fig. 2), what is in contrast to C. albicans- or CMV-specific cells and hence rather speaks against gut hom- ing as major contributor for depleting the pool of bacteria-specific CD4+ T cells from the circulation. Unfortunately, we did not have the chance to investigate gut biopsies from these patients to cor- roborate this statement. Since IBD patients were treated with anti- TNF-α (Infliximab) that was reported to downregulate INF-γ pro- duction by T cells [28, 29], we cannot exclude that we underesti- mated the frequencies of gut microbe-specific CD4+ T cells based on their ability to produce IFN-γ upon in vitro restimulation. Also, repetitive stimulation of gut-microbe-specific CD4+ T cells by bac- terial antigens in the context of microbial translocation [30] could induce their functional impairment that might explain why those cells cannot be detected upon restimulation with the correspond- ing bacterial lysates.

Concluding remarks

In summary, our study shows that there are low frequencies of gut microbe-specific CD4+ T cells in the periphery of HIV-1 patients and there are likely a number of factors precluding accumula- tion of those cells in circulation despite microbial translocation and enteropathy. However, we can conclude that gut commensal microbe specificities are not effectively contributing to the periph- eral population of activated CD4+ T cells and thereby our study excludes gut commensal derived cognate antigens as major driving force for TCR-dependent CD4+ T-cell activation.

Materials and methods

Patients

HIV patients (N = 22) were enrolled in the Zurich Primary HIV Infection Study (www.clinicaltrials.gov; ID: NCT00537966) [31] who initiated cART treatment within 90 days after acute infection. Patients chosen for our study experienced an interruption of ther- apy for at least 1 year, cART had been resumed on average 6 years before blood samples were drawn for the present study and no viral replication was detectable at that time point [32, 33]. Patients with clinically and histologically documented IBD (N = 19) were enrolled by the Swiss IBD cohort study (http://ibdcohort.ch), only patients receiving Infliximab (anti-TNF-α) were included in this study. Patients on immunosuppressive therapies were excluded.

Healthy individuals of comparable age were recruited at the same time. Approval and written informed consent from all patients were obtained according to the guidelines of the Ethics Committee of the University Hospital Zurich and the Triemli Hos- pital.

Bacterial and Candida lysate

Overnight cultures of E. coli, K. pneumoniae, and E. faecalis (pri- mary isolates from stool samples of healthy individuals), B. thetaio- taomicron (gift from Dr. Emma Slack), B. japonicum (gift from Prof. Dr. Hauke Hennecke), and C. albicans strain SC5314 [34] (gift from Prof. Dr. Salomé Leibundgut-Landmann) were adjusted to 1 × 108 cells/mL in PBS. Bacteria were sonicated and incu- bated on ice alternately for 1 min, C. albicans was boiled for at least 1 h. Lysate concentration used for the stimulation of CD4+

T cells corresponded to 1 × 105 cells/mL. Lysates were stored frozen at –20°C or kept at 4°C if used within a couple of weeks.

Enumeration of cytokine producing CD4+ T cells

CD4+ T cells and CD14+ monocytes were isolated from PBMCs using human CD4 and -CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instruction. CD4+ T cells were stimulated with bacterial and can- dida lysates or 15-mer peptides covering the entire immunodomi- nant CMV pp65 protein or IE-1 in the presence of freshly isolated autologous CD14+ monocytes in a ratio of 4 to 1 for 18 h at 37°C. A total of 2 – 2.5 × 105 CD4+ cells were used in enzyme- linked immuno spot (ELISpot) assays, whereas 1 × 106 CD4+ cells were used for analysis by FACS. MHCII-specific Ab (clone TÜ39; f.c. 2.5 μg/mL, BioLegend, Switzerland) or brefeldin A (f.c. 10 μg/mL, Sigma, Switzerland) was added if required. For assay controls, CD4+ T cells were left unstimulated or stimulated with PMA & ionomycin (50 and 500 ng/mL, Sigma, Switzer- land). The assay was performed in duplicates in a total volume of 200 μL of RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine (all reagents from PAA Laboratories, Austria). Detection of antigen- specific CD4+ T-cell responses by ELISpot (Mabtech, Sweden) was performed according to the manufacturer’s instructions using MAIPSWU plates (Millipore, Darmstadt, Germany) and spots were counted on an AID ELISpot reader (CTL, Germany). For the anal- ysis of intracellular IL-17 and IFN-γ expression, cells were per- meabilized after surface staining with Permeabilization buffer (BioLegend, San Diego, USA) and stained for 30 min at RT with fluorchrome conjugated monoclonal antibodies (anti-IL-17: clone BL168; anti-IFN-γ: clone B27). Surface staining was performed with fluorchrome conjugated monoclonal anti-CD3 (clone UHCT- 1), anti-CD4 (clone SK3), anti-CD38 (clone HIT-2a), anti-HLA-DR (clone L243), anti-integrin β antibody (clone FIB504), and CD161 (clone HP-3G10) for 30 min at 4°C. LIVE/DEAD cell stain (Life Technologies, Carlsbad, USA) was used for the exclusion of dead

3112 Kathrin Zimmermann et al. Eur. J. Immunol. 2015. 45: 3107–3113

cells. LSR II and FACSDiva software (BD Biosciences, Franklin Lakes, USA) were used for the acquisition of flow cytometry data, and FlowJo software (TreeStar) was used for analysis.

All antibodies were purchased from BD Biosciences, Switzer- land, or BioLegend, San Diego, USA). Data were analyzed and plotted with GraphPad Prism (GraphPad, La Jolla, CA, USA) using scatter plots with bars representing median values.

Acknowledgments: We are grateful to the patients who agreed to donate blood to support our experiments and we especially thank the study nurses and doctors in the Triemli hospital as well as at the department of infectious disease at the University hospital in Zurich for their extra efforts. A special thank goes to Dr. med Dominique Braun and Christina Grube for excellent patient care within the ZPHI study. We are grateful to Dr. Peter Mirtschink who took blood from healthy donors. We thank Dr. Hauke Hennecke for providing us with B japonicum and Dr. Salomé LeibundGut- Landmann for providing us with C. albicans. This work was sup- ported by the ETH Zurich, the Swiss National Science Foundation (Grant No. 310030 129751 to A.O., by the University of Zurich’s Clinical research Priority Program (CRPP) “Viral infectious dis- eases: Zurich Primary HIV Infection Study” to H.F.G.) and the Horten Foundation. Thanks to the Institute of Medical Microbiol- ogy, University of Zurich, for the isolation and typefication of gut commensal bacteria derived from primary stool samples.

Conflict of interest: The authors declare no commercial or finan- cial conflict of interest.

References

1 Appay, V. and Sauce, D., Immune activation and inflammation in HIV-1

infection: causes and consequences. J. Pathol. 2008. 214: 231–241.

2 Giorgi, J. V., Hultin, L. E., McKeating, J. A., Johnson, T. D., Owens, B.,

Jacobson, L. P., Shih, R. et al., Shorter survival in advanced human

immunodeficiency virus type 1 infection is more closely associated

with T lymphocyte activation than with plasma virus burden or virus

chemokine coreceptor usage. J. Infect Dis. 1999. 179: 859–870.

3 Beignon, A. S., McKenna, K., Skoberne, M., Manches, O., DaSilva, I.,

Kavanagh, D. G., Larsson, M. et al., Endocytosis of HIV-1 activates plas-

macytoid dendritic cells via Toll-like receptor-viral RNA interactions. J.

Clin. Invest. 2005. 115: 3265–3275.

4 Douek, D. C., Brenchley, J. M., Betts, M. R., Ambrozak, D. R., Hill, B. J.,

Okamoto, Y., Casazza, J. P. et al., HIV preferentially infects HIV-specific

CD4+ T cells. Nature 2002. 417: 95–98. 5 Harper, M. E., Marselle, L. M., Gallo, R. C. and Wong-Staal, F., Detec-

tion of lymphocytes expressing human T-lymphotropic virus type III in

lymph nodes and peripheral blood from infected individuals by in situ

hybridization. Proc. Natl. Acad. Sci. USA 1986. 83: 772–776.

6 Kaiser, P., Joos, B., Niederost, B., Weber, R., Gunthard, H. F. and Fis-

cher, M., Productive human immunodeficiency virus type 1 infection in

peripheral blood predominantly takes place in CD4/CD8 double-negative

T lymphocytes. J. Virol. 2007. 81: 9693–9706.

7 Virgin, H. W., Wherry, E. J. and Ahmed, R., Redefining chronic viral infec-

tion. Cell 2009. 138: 30–50.

8 Doisne, J. M., Urrutia, A., Lacabaratz-Porret, C., Goujard, C., Meyer, L.,

Chaix, M. L., Sinet, M. and Venet, A., CD8+ T cells specific for EBV, cytomegalovirus, and influenza virus are activated during primary HIV

infection. J. Immunol. 2004. 173: 2410–2418.

9 Brenchley, J. M., Price, D. A., Schacker, T. W., Asher, T. E., Silvestri, G.,

Rao, S., Kazzaz, Z. et al., Microbial translocation is a cause of systemic

immune activation in chronic HIV infection. Nat. Med. 2006. 12: 1365–

1371.

10 Brenchley, J. M., Schacker, T. W., Ruff, L. E., Price, D. A., Taylor, J. H.,

Beilman, G. J., Nguyen, P. L. et al., CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J. Exp.

Med. 2004. 200: 749–759.

11 Mehandru, S., Poles, M. A., Tenner-Racz, K., Horowitz, A., Hurley, A.,

Hogan, C., Boden, D. et al., Primary HIV-1 infection is associated with

preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. J. Exp. Med. 2004. 200: 761–770.

12 Bastidas, S., Graw, F., Smith, M. Z., Kuster, H., Gunthard, H. F. and

Oxenius, A., CD8+ T cells are activated in an antigen-independent manner in HIV-infected individuals. J. Immunol. 2014. 192: 1732–

1744.

13 Haas, A., Rehr, M., Graw, F., Rusert, P., Bossart, W., Kuster, H.,

Trkola, A. et al., HIV-1 replication activates CD4+ T cells with speci- ficities for persistent herpes viruses. EMBO Mol. Med. 2010. 2: 231–

244.

14 Haas, A., Zimmermann, K. and Oxenius, A., Antigen-dependent and -

independent mechanisms of T and B cell hyperactivation during chronic

HIV-1 infection. J. Virol. 2011. 85: 12102–12113.

15 Barron, M. A., Blyveis, N., Palmer, B. E., MaWhinney, S. and Wilson, C.

C., Influence of plasma viremia on defects in number and immunophe-

notype of blood dendritic cell subsets in human immunodeficiency virus

1-infected individuals. J. Infect Dis. 2003. 187: 26–37.

16 Dillon, S. M., Robertson, K. B., Pan, S. C., Mawhinney, S., Meditz, A. L.,

Folkvord, J. M., Connick, E. et al., Plasmacytoid and myeloid dendritic

cells with a partial activation phenotype accumulate in lymphoid tissue

during asymptomatic chronic HIV-1 infection. J. Acquir. Immune Defic.

Syndr. 2008. 48: 1–12.

17 Smith, M. Z., Bastidas, S., Karrer, U. and Oxenius, A., Impact of antigen

specificity on CD4+ T cell activation in chronic HIV-1 infection. BMC Infect Dis. 2013. 13: 100.

18 McGuckin, M. A., Eri, R., Simms, L. A., Florin, T. H. and Radford-Smith, G.,

Intestinal barrier dysfunction in inflammatory bowel diseases. Inflamm.

Bowel Dis. 2009. 15: 100–113.

19 Pastor Rojo, O., Lopez San Roman, A., Albeniz Arbizu, E., de la

Hera Martinez, A., Ripoll Sevillano, E. and Albillos Martinez, A.,

Serum lipopolysaccharide-binding protein in endotoxemic patients

with inflammatory bowel disease. Inflamm. Bowel Dis. 2007. 13: 269–

277.

20 Slack, E., Hapfelmeier, S., Stecher, B., Velykoredko, Y., Stoel, M., Lawson,

M. A., Geuking, M. B. et al., Innate and adaptive immunity cooperate

flexibly to maintain host-microbiota mutualism. Science 2009. 325: 617–

620.

21 Sedman, P. C., Macfie, J., Sagar, P., Mitchell, C. J., May, J., Mancey-Jones,

B. and Johnstone, D., The prevalence of gut translocation in humans.

Gastroenterology 1994. 107: 643–649.

Eur. J. Immunol. 2015. 45: 3107–3113 Immunity to infection 3113

22 Haas, A., Zimmermann, K., Graw, F., Slack, E., Rusert, P., Ledergerber,

B., Bossart, W. et al., Systemic antibody responses to gut commensal

bacteria during chronic HIV-1 infection. Gut 2011. 60: 1506–1519.

23 Zimmermann, K., Haas, A. and Oxenius, A., Systemic antibody responses

to gut microbes in health and disease. Gut Microbes 2012. 3: 42–47.

24 Hand, T. W., Dos Santos, L. M., Bouladoux, N., Molloy, M. J., Pagan,

A. J., Pepper, M., Maynard, C. L. et al., Acute gastrointestinal infection

induces long-lived microbiota-specific T cell responses. Science 2012. 337:

1553–1556.

25 Costiniuk, C. T. and Angel, J. B., Human immunodeficiency virus and

the gastrointestinal immune system: does highly active antiretroviral

therapy restore gut immunity? Mucosal Immunol. 2012. 5: 596–604.

26 Souza, H. S., Elia, C. C., Spencer, J. and MacDonald, T. T., Expression of

lymphocyte-endothelial receptor-ligand pairs, alpha4beta7/MAdCAM-1

and OX40/OX40 ligand in the colon and jejunum of patients with inflam-

matory bowel disease. Gut 1999. 45: 856–863.

27 Mavigner, M., Cazabat, M., Dubois, M., L’Faqihi, F. E., Requena, M.,

Pasquier, C., Klopp, P. et al., Altered CD4+ T cell homing to the gut impairs mucosal immune reconstitution in treated HIV-infected individ-

uals. J. Clin. Invest 2012. 122: 62–69.

28 Haider, A. S., Cohen, J., Fei, J., Zaba, L. C., Cardinale, I., Toyoko, K.,

Ott, J. and Krueger, J. G., Insights into gene modulation by therapeutic

TNF and IFNgamma antibodies: TNF regulates IFNgamma production

by T cells and TNF-regulated genes linked to psoriasis transcriptome. J.

Invest Dermatol. 2008. 128: 655–666.

29 Agnholt, J. and Kaltoft, K., Infliximab downregulates interferon-gamma

production in activated gut T-lymphocytes from patients with Crohn’s

disease. Cytokine 2001. 15: 212–222.

30 Perreau, M., Vigano, S., Bellanger, F., Pellaton, C., Buss, G., Comte, D.,

Roger, T. et al., Exhaustion of bacteria-specific CD4 T cells and microbial

translocation in common variable immunodeficiency disorders. J. Exp.

Med. 2014. 211: 2033–2045.

31 Rieder, P., Joos, B., Scherrer, A. U., Kuster, H., Braun, D., Grube, C.,

Niederost, B. et al., Characterization of human immunodeficiency virus

type 1 (HIV-1) diversity and tropism in 145 patients with primary HIV-1

infection. Clin. Infect Dis. 2011. 53: 1271–1279.

32 Wyl, V., Gianella, S., Fischer, M., Niederoest, B., Kuster, H., Battegay,

M., Bernasconi, E. et al., Early antiretroviral therapy during primary HIV-

1 infection results in a transient reduction of the viral setpoint upon

treatment interruption. PLoS One 2011. 6: e27463.

33 Gianella, S., von Wyl, V., Fischer, M., Niederoest, B., Battegay, M.,

Bernasconi, E., Cavassini, M. et al., Effect of early antiretroviral ther-

apy during primary HIV-1 infection on cell-associated HIV-1 DNA and

plasma HIV-1 RNA. Antivir. Ther. 2011. 16: 535–545.

34 Fonzi, W. A. and Irwin, M. Y., Isogenic strain construction and gene

mapping in Candida albicans. Genetics 1993. 134: 717–728.

Abbreviations: cART: antiretroviral combination therapy · ELISpot: enzyme-linked immuno spot assay · GIT: gastrointestinal tract · IBD: Inflammatory bowel disease

Full correspondence: Prof. Annette Oxenius, Institute of Microbiology, Swiss Federal Institute of Technology Zurich, Vladimir-Prelog-Weg 1–5/10, HCI G401, 8093 Zurich, Switzerland Fax: +41-44-632-10-98 e-mail: [email protected]

Received: 20/7/2015 Revised: 20/8/2015 Accepted: 8/9/2015