B cell follicle sanctuary permits persistent productive simian immunodeficiency virus infection in elite controllers

Journal name:
Nature Medicine
Volume:
21,
Pages:
132–139
Year published:
DOI:
doi:10.1038/nm.3781
Received
Accepted
Published online

Abstract

Chronic-phase HIV and simian immunodeficiency virus (SIV) replication is reduced by as much as 10,000-fold in elite controllers (ECs) compared with typical progressors (TPs), but sufficient viral replication persists in EC tissues to allow viral sequence evolution and induce excess immune activation. Here we show that productive SIV infection in rhesus monkey ECs, but not TPs, is markedly restricted to CD4+ follicular helper T (TFH) cells, suggesting that these EC monkeys' highly effective SIV-specific CD8+ T cells can effectively clear productive SIV infection from extrafollicular sites, but their relative exclusion from B cell follicles prevents their elimination of productively infected TFH cells. CD8+ lymphocyte depletion in EC monkeys resulted in a dramatic re-distribution of productive SIV infection to non-TFH cells, with restriction of productive infection to TFH cells resuming upon CD8+ T cell recovery. Thus, B cell follicles constitute 'sanctuaries' for persistent SIV replication in the presence of potent anti-viral CD8+ T cell responses, potentially complicating efforts to cure HIV infection with therapeutic vaccination or T cell immunotherapy.

At a glance

Figures

  1. The distribution of productive SIV infection within CD4+ memory T cell subsets differs in chronic-phase, but not acute-phase, attenuated versus WT SIVmac239 infection.
    Figure 1: The distribution of productive SIV infection within CD4+ memory T cell subsets differs in chronic-phase, but not acute-phase, attenuated versus WT SIVmac239 infection.

    (a) Plasma VL profiles of WT SIVmac239 and attenuated SIVmac239Δnef infections in the indicated monkeys. (b,c) Flow cytometric analysis of intracellular SIV-Gag p27 to detect replication-competent SIV within PD-1– and CD200–defined CD4+ memory T cell populations (TFH versus non-TFH cells; 104 sorted cells, isolated as shown in Supplementary Fig. 1). Measurements obtained at designated time points after infection with WT SIVmac239 (Rh27617; b) and attenuated SIVmac239Δnef (Rh25653; c) after 17 d of coculture with CEMx174 cells; percentages of Gag+ cells are shown. Identical results were observed for Rh26310 (WT SIVmac239) and Rh25714 (SIVmac239Δnef)(Supplementary Fig. 3).

  2. The distribution of productive WT SIV infection within CD4+ memory T cells in LN correlates with immune control.
    Figure 2: The distribution of productive WT SIV infection within CD4+ memory T cells in LN correlates with immune control.

    (a) Representative flow cytometric profiles (n = 7–17 samples per group; see b) showing the frequencies (percentage) of PD-1– and CD200–defined subsets within the (CD95high) CD4+ memory T cell populations in LNs of SIVneg, elite controller (EC), semi-controller (Semi), progressor (Prog), and cART-suppressed SIV-infected (cART) monkeys (see Supplementary Tables 1 and 2). (b) Relative frequencies of CD4+ TFH cells (CD200highPD-1high) within the total CD4+ memory T cell populations in LNs of the designated monkey groups (black bars indicate the median value for each group). The Kruskal-Wallis test determined the significance of overall differences in the percentage of CD4+ TFH cells among these monkey groups. P value shown, and if this P value was <0.05, the Wilcoxon rank-sum test was used to perform pairwise analysis (brackets indicate P < 0.05). (c,d) Detection of replication-competent SIVmac239 from sorted PD-1– and CD200–defined CD4+ memory T cell subsets of LNs from EC and Semi monkeys after 13 d of CEMx174 cell coculture with 105 sorted LN cells, and from Prog monkeys after 17 d of CEMx174 cell coculture with 104 sorted LN cells (note that fewer sorted CD4+ memory T cells were available in progressor monkeys owing to CD4+ T cell depletion). Representative results are shown in c, with plasma viral load (in SIV RNA copies per ml) at the time of biopsy shown in parenthesis under the monkey identification numbers, and data from all analyses are shown in d (n = 5–7 for all groups) with light colored lines delineating individual monkeys and bold diamond symbols delineating log median values of each group. The Friedman test determined the significance of overall differences in replication-competent SIV among the PD-1– and CD200–defined CD4+ memory T cell subsets (P values shown; NS, not significant). The Wilcoxon signed-rank test was used for pairwise analysis when the Friedman P value was <0.05 (brackets indicate Wilcoxon signed-rank test, P < 0.05). (e,f) Comparison of cell-associated SIV RNA (e) and DNA (f) levels in the same LN CD4+ memory T cell subsets (n = 7–10 for all groups), with statistical analysis as described in d.

  3. Productive SIV infection in monkeys is anatomically restricted to B cell follicles in EC, but not TP, monkeys.
    Figure 3: Productive SIV infection in monkeys is anatomically restricted to B cell follicles in EC, but not TP, monkeys.

    (a,b) Representative SIV (red) in situ hybridization images from an EC monkey (Rh24827; a; of n = 6 examined) versus a chronically SIV–infected progressor (Rh-P383; b; of n = 6 examined). Yellow boxes indicate areas shown at higher magnification in the inset (a) or in a separate image (b, right). B cell follicles (F) are demarcated with dotted lines. Black arrows indicate all SIV RNA+ lymphoid cells outside of follicles and within the paracortical T cell zone (TZ). Black arrowheads point to all SIV RNA+ lymphoid cells within B cell follicles (note intense cell-centric SIV RNA signal within a single cell, consistent with productive infection). These productively infected cells are distinct from the well-characterized lattice-like filamentous pattern of extracellular follicular dendritic cell–bound virus that is also present within follicles (white arrowheads)31. Black scale bars, 100 μm and 60 μm in low- and high-magnification images, respectively. (c) Quantification of the percentage of total SIV RNA+ cells within the LN of EC versus TP monkeys that are within B cell follicles (bars indicate median values). The Wilcoxon rank-sum test determined the significance of differences in this percentage (P value shown). (d) Representative confocal micrograph of a LN section from an EC monkey (Rh24827; of n = 5 examined) showing conventional in situ hybridization for SIV RNA (green) in combination with CD20- (white) and CD8-specific (red) antibody staining; the yellow box at left image indicates the area shown at higher magnification at right. Yellow arrows indicate all productively SIV-infected cells in the image. Yellow scale bars, 200 μm and 60 μm in low- and high-magnification images, respectively.

  4. The restriction of productive SIV infection to CD4+ TFH cells in EC LNs is lost with in vivo CD8+ lymphocyte depletion.
    Figure 4: The restriction of productive SIV infection to CD4+ TFH cells in EC LNs is lost with in vivo CD8+ lymphocyte depletion.

    (ac) Effect of CD8+ lymphocyte depletion on plasma VL and on frequencies of total CD3+CD8+ T cells (a), SIV-specific CD8+ T cells (identified by intracellular cytokine analysis; b), and total CD3−CD8+ NK cells (c) in peripheral blood and LNs of 7 EC monkeys (Supplementary Table 1; note that one monkey, Rh27033, was taken to necropsy at day 10, reducing n to 6 at later time points). Error bars indicate mean + sem. (d) Detection of replication-competent SIV from the designated sorted LN CD4+ memory T cell populations (105 cells cocultured with CEMx174 for 17 d) from a representative EC monkey (Rh26623) of the 7 studied before and after CD8-specific antibody treatment. (e,f) Analysis of the distribution of replication-competent SIV by CEMx174 coculture assay (e; 105 sorted LN cells; 17–22 d) and cell-associated SIV RNA and DNA levels by RT-PCR and PCR, respectively (f), among LN (PD-1 and CD200 defined) CD4+ memory T cell subsets before and after CD8-specific antibody treatment of seven ECs (Supplementary Table 1). Light-colored lines, individual monkeys; bold diamonds, log median values of each group. Statistical analysis was performed as described in Figure 2d; brackets indicate Wilcoxon signed-rank test; P < 0.05.

  5. The restriction of productive SIV infection to CD4+ TFH cells in EC LNs is not affected by activation of extrafollicular CD4+ memory T cells with IL-7 administration.
    Figure 5: The restriction of productive SIV infection to CD4+ TFH cells in EC LNs is not affected by activation of extrafollicular CD4+ memory T cells with IL-7 administration.

    Three EC monkeys that had recovered both CD8+ T cell counts and CD8+ T cell–mediated SIV control (plasma VL of <100 copies per milliliter) 5–6 months after CD8-specific antibody treatment were treated with recombinant rhesus IL-7 to activate extrafollicular CD4+ memory T cells. (a) Comparison of the induction of the proliferation marker Ki-67 on LN non-TFH cells (PD-1neg/dimCD200neg/dim) CD4+ memory T cells from these three EC monkeys after CD8+ lymphocyte depletion (black lines) versus after IL-7 treatment (red lines; percentage change of Ki-67 = after-treatment percentage of Ki-67+ – baseline percentage of Ki-67+). Each symbol represents a different monkey (square = Rh25687; triangle = Rh26623; circle = Rh25610). (b) Comparison of plasma VL after CD8+ lymphocyte depletion with plasma VL after IL-7 treatment (same monkeys as in a). (c) Detection of replication-competent SIV by CEMx174 coculture analysis from sorted LN (PD-1– and CD200–defined) CD4+ memory T subsets of all three IL-7-treated EC monkeys 10 d after IL-7 treatment (the peak of non-TFH CD4+ memory T cell proliferation). Contrast the effect of IL-7 treatment on the distribution of replication-competent SIV among CD4+ TFH versus non- TFH memory cells (day 10 after treatment) in this figure with that of CD8+ lymphocyte depletion at the same day-10-post-treatment time point in Figure 4d,e.

  6. CD4+ TFH cells contain higher levels of cell-associated SIV RNA than non-TFH CD4+ memory T cells in SIV+ monkeys with effective suppression of viral replication.
    Figure 6: CD4+ TFH cells contain higher levels of cell-associated SIV RNA than non-TFH CD4+ memory T cells in SIV+ monkeys with effective suppression of viral replication.

    (a,b) Comparison of the levels of cell-associated SIV RNA and DNA in sorted CD4+ memory T cell subsets (PD-1neg and PD-1dim non-TFH vs. PD-1high TFH cells; Supplementary Fig. 1) from LN (a) and spleen (b) of 7 cohort 1 monkeys, which were chronically SIVmac251-infected before cART treatment; Supplementary Table 2). (c) Levels of cell-associated SIV RNA and DNA in sorted CD4+ memory T cell subsets (TCM and TTr/EM cells versus TFH cells; Supplementary Fig. 8) from LN of ten cohort 2 monkeys, which were treated with cART 42 d after infection with SIVmac239 (Supplementary Table 2). Light-colored lines, individual monkeys; bold diamonds, log median values of each group. Statistical analysis was performed as described in Figure 2d; brackets indicate Wilcoxon signed-rank test, P < 0.05.

References

  1. Kirchhoff, F. Immune evasion and counteraction of restriction factors by HIV-1 and other primate lentiviruses. Cell Host Microbe 8, 5567 (2010).
  2. Picker, L.J., Hansen, S.G. & Lifson, J.D. New paradigms for HIV/AIDS vaccine development. Annu. Rev. Med. 63, 95111 (2012).
  3. Okoye, A.A. & Picker, L.J. CD4+ T cell depletion in HIV infection: mechanisms of immunological failure. Immunol. Rev. 254, 5464 (2013).
  4. Deeks, S.G. & Walker, B.D. Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy. Immunity 27, 406416 (2007).
  5. Mudd, P.A. & Watkins, D.I. Understanding animal models of elite control: windows on effective immune responses against immunodeficiency viruses. Curr. Opin. HIV AIDS 6, 197201 (2011).
  6. Katlama, C. et al. Barriers to a cure for HIV: new ways to target and eradicate HIV-1 reservoirs. Lancet 381, 21092117 (2013).
  7. Hatano, H. et al. Evidence for persistent low-level viremia in individuals who control human immunodeficiency virus in the absence of antiretroviral therapy. J. Virol. 83, 329335 (2009).
  8. Pereyra, F. et al. Persistent low-level viremia in HIV-1 elite controllers and relationship to immunologic parameters. J. Infect. Dis. 200, 984990 (2009).
  9. Chun, T.W. et al. Effect of antiretroviral therapy on HIV reservoirs in elite controllers. J. Infect. Dis. 208, 14431447 (2013).
  10. Bailey, J.R., Williams, T.M., Siliciano, R.F. & Blankson, J.N. Maintenance of viral suppression in HIV-1-infected HLA-B*57+ elite suppressors despite CTL escape mutations. J. Exp. Med. 203, 13571369 (2006).
  11. Mens, H. et al. HIV-1 continues to replicate and evolve in patients with natural control of HIV infection. J. Virol. 84, 1297112981 (2010).
  12. O'Connell, K.A. et al. Control of HIV-1 in elite suppressors despite ongoing replication and evolution in plasma virus. J. Virol. 84, 70187028 (2010).
  13. Hunt, P.W. et al. Relationship between T cell activation and CD4+ T cell count in HIV-seropositive individuals with undetectable plasma HIV RNA levels in the absence of therapy. J. Infect. Dis. 197, 126133 (2008).
  14. Hatano, H. et al. Prospective antiretroviral treatment of asymptomatic, HIV-1 infected controllers. PLoS Pathog. 9, e1003691 (2013).
  15. Walker, B.D. & Yu, X.G. Unravelling the mechanisms of durable control of HIV-1. Nat. Rev. Immunol. 13, 487498 (2013).
  16. Sáez-Cirión, A. & Pancino, G. HIV controllers: a genetically determined or inducible phenotype? Immunol. Rev. 254, 281294 (2013).
  17. Mendoza, D. et al. Cytotoxic capacity of SIV-specific CD8+ T cells against primary autologous targets correlates with immune control in SIV-infected rhesus macaques. PLoS Pathog. 9, e1003195 (2013).
  18. Mudd, P.A. et al. Reduction of CD4+ T cells in vivo does not affect virus load in macaque elite controllers. J. Virol. 85, 74547459 (2011).
  19. Friedrich, T.C. et al. Subdominant CD8+ T cell responses are involved in durable control of AIDS virus replication. J. Virol. 81, 34653476 (2007).
  20. Blankson, J.N. et al. Isolation and characterization of replication-competent human immunodeficiency virus type 1 from a subset of elite suppressors. J. Virol. 81, 25082518 (2007).
  21. Fukazawa, Y. et al. Lymph node T cell responses predict the efficacy of live attenuated SIV vaccines. Nat. Med. 18, 16731681 (2012).
  22. Crotty, S. Follicular helper CD4 T cells (TFH). Annu. Rev. Immunol. 29, 621663 (2011).
  23. Vinuesa, C.G. & Cyster, J.G. How T cells earn the follicular rite of passage. Immunity 35, 671680 (2011).
  24. Connick, E. et al. CTL fail to accumulate at sites of HIV-1 replication in lymphoid tissue. J. Immunol. 178, 69756983 (2007).
  25. Tjernlund, A. et al. In situ detection of Gag-specific CD8+ cells in the GI tract of SIV infected Rhesus macaques. Retrovirology 7, 114 (2010).
  26. Sasikala-Appukuttan, A.K. et al. Location and dynamics of the immunodominant CD8 T cell response to SIVnef immunization and SIVmac251 vaginal challenge. PLoS ONE 8, e81623 (2013).
  27. Robinson, H.L. & Amara, R.R. Protective immunity from a germinal center sanctuary. Nat. Med. 18, 16141616 (2012).
  28. Skinner, P.J. & Connick, E. Overcoming the immune privilege of B cell follicles to cure HIV-1 infection. J. Hum. Virol. Retrovirol. 1, 0000100003. doi:jhvrv.2014.01.00001 (2014).
  29. Perreau, M. et al. Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production. J. Exp. Med. 210, 143156 (2013).
  30. Lindqvist, M. et al. Expansion of HIV-specific T follicular helper cells in chronic HIV infection. J. Clin. Invest. 122, 32713280 (2012).
  31. Brenchley, J.M. et al. Differential infection patterns of CD4+ T cells and lymphoid tissue viral burden distinguish progressive and nonprogressive lentiviral infections. Blood 120, 41724181 (2012).
  32. Petrovas, C. et al. CD4 T follicular helper cell dynamics during SIV infection. J. Clin. Invest. 122, 32813294 (2012).
  33. Klatt, N.R. et al. SIV infection of rhesus macaques results in dysfunctional T and B cell responses to neo and recall Leishmania major vaccination. Blood 118, 58035812 (2011).
  34. Chtanova, T. et al. T follicular helper cells express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells. J. Immunol. 173, 6878 (2004).
  35. Estes, J.D. Pathobiology of HIV/SIV-associated changes in secondary lymphoid tissues. Immunol. Rev. 254, 6577 (2013).
  36. Okoye, A. et al. Profound CD4+/CCR5+ T cell expansion is induced by CD8+ lymphocyte depletion but does not account for accelerated SIV pathogenesis. J. Exp. Med. 206, 15751588 (2009).
  37. Leone, A. et al. Increased CD4+ T cell levels during IL-7 administration of antiretroviral therapy-treated simian immunodeficiency virus-positive macaques are not dependent on strong proliferative responses. J. Immunol. 185, 16501659 (2010).
  38. Quigley, M.F., Gonzalez, V.D., Granath, A., Andersson, J. & Sandberg, J.K. CXCR5+ CCR7 CD8 T cells are early effector memory cells that infiltrate tonsil B cell follicles. Eur. J. Immunol. 37, 33523362 (2007).
  39. Wood, G.S., Garcia, C.F., Dorfman, R.F. & Warnke, R.A. The immunohistology of follicle lysis in lymph node biopsies from homosexual men. Blood 66, 10921097 (1985).
  40. Tenner-Rácz, K. et al. Monoclonal antibodies to human immunodeficiency virus: their relation to the patterns of lymph node changes in persistent generalized lymphadenopathy and AIDS. AIDS 1, 95104 (1987).
  41. Tenner-Racz, K. et al. Cytotoxic effector cell granules recognized by the monoclonal antibody TIA-1 are present in CD8+ lymphocytes in lymph nodes of human immunodeficiency virus-1-infected patients. Am. J. Pathol. 142, 17501758 (1993).
  42. Petrovas, C. et al. A population of CD8+ T cells located in germinal centers that is functionally capable of mediating bispecific antibody killing of HIV-infected T cells. 20th International AIDS Conference Melbourne, Australia, http://pag.aids2014.org/Abstracts.aspx?SID=1147&AID=5387 (2014).
  43. Shan, L. et al. Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation. Immunity 36, 491501 (2012).
  44. Anolik, J.H. & Aringer, M. New treatments for SLE: cell-depleting and anti-cytokine therapies. Best Pract. Res. Clin. Rheumatol. 19, 859878 (2005).
  45. Hansen, S.G. et al. Immune clearance of highly pathogenic SIV infection. Nature 502, 100104 (2013).
  46. Cline, A.N., Bess, J.W., Piatak, M. Jr. & Lifson, J.D. Highly sensitive SIV plasma viral load assay: practical considerations, realistic performance expectations, and application to reverse engineering of vaccines for AIDS. J. Med. Primatol. 34, 303312 (2005).
  47. Venneti, S. et al. Longitudinal in vivo positron emission tomography imaging of infected and activated brain macrophages in a macaque model of human immunodeficiency virus encephalitis correlates with central and peripheral markers of encephalitis and areas of synaptic degeneration. Am. J. Pathol. 172, 16031616 (2008).
  48. Brown, C.R. et al. Unique pathology in simian immunodeficiency virus-infected rapid progressor macaques is consistent with a pathogenesis distinct from that of classical AIDS. J. Virol. 81, 55945606 (2007).
  49. Smedley, J. et al. Tracking the luminal exposure and lymphatic drainage pathways of intravaginal and intrarectal inocula used in nonhuman primate models of HIV transmission. PLoS ONE 9, e92830 (2014).
  50. Wolfe, M.H.D.A. Nonparametric Statistical Methods (John Wiley & Sons, New York, 1999).
  51. R Core Team. R: a language and environment for statistical computing. (http://www.r-project.org/) (R Foundation for Statistical Computing, Vienna, Austria, 2014).

Download references

Author information

Affiliations

  1. Vaccine and Gene Therapy Institute, Oregon Health & Science University, Beaverton, Oregon, USA.

    • Yoshinori Fukazawa,
    • Richard Lum,
    • Afam A Okoye,
    • Haesun Park,
    • Jin Young Bae,
    • Shoko I Hagen,
    • Tonya Swanson,
    • Alfred W Legasse,
    • Michael K Axthelm &
    • Louis J Picker
  2. Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, Oregon, USA.

    • Yoshinori Fukazawa,
    • Richard Lum,
    • Afam A Okoye,
    • Haesun Park,
    • Jin Young Bae,
    • Shoko I Hagen,
    • Tonya Swanson,
    • Alfred W Legasse,
    • Michael K Axthelm &
    • Louis J Picker
  3. Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

    • Kenta Matsuda &
    • Vanessa M Hirsch
  4. AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, Maryland, USA.

    • Rebecca Shoemaker,
    • Claire Deleage,
    • Carissa Lucero,
    • David Morcock,
    • Michael Piatak Jr,
    • Jacob D Estes &
    • Jeffrey D Lifson
  5. Gilead Sciences, Inc., Foster City, California, USA.

    • Joseph Hesselgesser &
    • Romas Geleziunas
  6. Statistical Center for HIV/AIDS Research and Prevention, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

    • Paul T Edlefsen

Contributions

L.J.P. and Y.F. conceived the study, designed the experiments and wrote the paper, assisted by A.A.O., H.P. and J.D.L. Y.F. also supervised the experiments, performed immunologic and SIV coculture assays, and analyzed the data, assisted by R.L., J.Y.B. and S.I.H. A.W.L. and M.K.A. managed the animal protocols, assisted by T.S. K.M., V.M.H., C.D., C.L., D.M. and J.D.E. provided tissue-based analysis including immunohistochemistry and RNAscope. M.P., Jr. and J.D.L. planned and performed SIV quantification, assisted by R.S. J.H. and R.G. developed and provided optimized cART regimens (cohort 2). P.T.E. performed the statistical analysis of study data.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (31,759 KB)

    Combined Supplementary Figures 1–9 and Supplementary Tables 1–2.

Additional data