Andrew W. Sylwester

Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects

Human cytomegalovirus (HCMV) infections of immunocompetent hosts are characterized by a dynamic, life-long interaction in which host immune responses, particularly of T cells, restrain viral replication and prevent disease but do not eliminate the virus or preclude transmission. Because HCMV is among the largest and most complex of known viruses, the T cell resources committed to maintaining this balance have never been characterized completely. Here, using cytokine flow cytometry and 13,687 overlapping 15mer peptides comprising 213 HCMV open reading frames (ORFs), we found that 151 HCMV ORFs were immunogenic for CD4+ and/or CD8+ T cells, and that ORF immunogenicity was influenced only modestly by ORF expression kinetics and function. We further documented that total HCMV-specific T cell responses in seropositive subjects were enormous, comprising on average ∼10% of both the CD4+ and CD8+ memory compartments in blood, whereas cross-reactive recognition of HCMV proteins in seronegative individuals was limited to CD8+ T cells and was rare. These data provide the first glimpse of the total human T cell response to a complex infectious agent and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans.

Insufficient Production and Tissue Delivery of CD4+Memory T Cells in Rapidly Progressive Simian Immunodeficiency Virus Infection

The mechanisms linking human immunodeficiency virus replication to the progressive immunodeficiency of acquired immune deficiency syndrome are controversial, particularly the relative contribution of CD4+ T cell destruction. Here, we used the simian immunodeficiency virus (SIV) model to investigate the relationship between systemic CD4+ T cell dynamics and rapid disease progression. Of 18 rhesus macaques (RMs) infected with CCR5-tropic SIVmac239 (n = 14) or CXCR4-tropic SIVmac155T3 (n = 4), 4 of the former group manifested end-stage SIV disease by 200 d after infection. In SIVmac155T3 infections, naive CD4+ T cells were dramatically depleted, but this population was spared by SIVmac239, even in rapid progressors. In contrast, all SIVmac239-infected RMs demonstrated substantial systemic depletion of CD4+ memory T cells by day 28 after infection. Surprisingly, the extent of CD4+ memory T cell depletion was not, by itself, a strong predictor of rapid progression. However, in all RMs destined for stable infection, this depletion was countered by a striking increase in production of short-lived CD4+ memory T cells, many of which rapidly migrated to tissue. In all rapid progressors (P < 0.0001), production of these cells initiated but failed by day 42 of infection, and tissue delivery of new CD4+ memory T cells ceased. Thus, although profound depletion of tissue CD4+ memory T cells appeared to be a prerequisite for early pathogenesis, it was the inability to respond to this depletion with sustained production of tissue-homing CD4+ memory T cells that best distinguished rapid progressors, suggesting that mechanisms of the CD4+ memory T cell generation play a crucial role in maintaining immune homeostasis in stable SIV infection.

Progressive CD4+ central–memory T cell decline results in CD4+ effector–memory insufficiency and overt disease in chronic SIV infection

Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4+ CCR5+ effector–memory T (TEM) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4+ memory T cell proliferation appears to prevent collapse of effector site CD4+ TEM cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4+ TEM cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4+ TEM cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4+ TEM cells from central–memory T (TCM) cell precursors. The instability of effector site CD4+ TEM cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5− CD4+ TCM cells. These data suggest that although CD4+ TEM cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4+ TCM cells.

Lymph node T cell responses predict the efficacy of live attenuated SIV vaccines.

Live attenuated simian immunodeficiency virus (SIV) vaccines (LAVs) remain the most efficacious of all vaccines in nonhuman primate models of HIV and AIDS, yet the basis of their robust protection remains poorly understood. Here we show that the degree of LAV-mediated protection against intravenous wild-type SIVmac239 challenge strongly correlates with the magnitude and function of SIV-specific, effector-differentiated T cells in the lymph node but not with the responses of such T cells in the blood or with other cellular, humoral and innate immune parameters. We found that maintenance of protective T cell responses is associated with persistent LAV replication in the lymph node, which occurs almost exclusively in follicular helper T cells. Thus, effective LAVs maintain lymphoid tissue-based, effector-differentiated, SIV-specific T cells that intercept and suppress early wild-type SIV amplification and, if present in sufficient frequencies, can completely control and perhaps clear infection, an observation that provides a rationale for the development of safe, persistent vectors that can elicit and maintain such responses.

Profound CD4+/CCR5+ T cell expansion is induced by CD8+ lymphocyte depletion but does not account for accelerated SIV pathogenesis

Depletion of CD8+ lymphocytes during acute simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) results in irreversible prolongation of peak-level viral replication and rapid disease progression, consistent with a major role for CD8+ lymphocytes in determining postacute-phase viral replication set points. However, we report that CD8+ lymphocyte depletion is also associated with a dramatic induction of proliferation among CD4+ effector memory T (TEM) cells and, to a lesser extent, transitional memory T (TTrM) cells, raising the question of whether an increased availability of optimal (activated/proliferating), CD4+/CCR5+ SIV “target” cells contributes to this accelerated pathogenesis. In keeping with this, depletion of CD8+ lymphocytes in SIV− RMs led to a sustained increase in the number of potential CD4+ SIV targets, whereas such depletion in acute SIV infection led to increased target cell consumption. However, we found that the excess CD4+ TEM cell proliferation of CD8+ lymphocyte–depleted, acutely SIV-infected RMs was completely inhibited by interleukin (IL)-15 neutralization, and that this inhibition did not abrogate the rapidly progressive infection in these RMs. Moreover, although administration of IL-15 during acute infection induced robust CD4+ TEM and TTrM cell proliferation, it did not recapitulate the viral dynamics of CD8+ lymphocyte depletion. These data suggest that CD8+ lymphocyte function has a larger impact on the outcome of acute SIV infection than the number and/or activation status of target cells available for infection and viral production.

Cytomegalovirus-Specific T Cell Immunity Is Maintained in Immunosenescent Rhesus Macaques

Although CMV infection is largely benign in immunocompetent people, the specific T cell responses associated with control of this persistent virus are enormous and must be maintained for life. These responses may increase with advanced age and have been linked to an “immune risk profile” that is associated with poor immune responsiveness and increased mortality in aged individuals. Based on this association, it has been suggested that CMV-specific T cell responses might become dysfunctional with age and thereby contribute to the development of immune senescence by homeostatic disruption of other T cell populations, diminished control of CMV replication, and/or excess chronic inflammation. In this study, we use the rhesus macaque (RM) model of aging to ask whether the quantity and quality of CMV-specific T cell responses differ between healthy adult RMs and elderly RMs that manifest hallmarks of immune aging. We demonstrate that the size of the CD4+ and CD8+ CMV-specific T cell pools are similar in adult versus old RMs and show essentially identical phenotypic and functional characteristics, including a dominant effector memory phenotype, identical patterns of IFN-γ, TNF-α, and IL-2 production and cytotoxic degranulation, and comparable functional avidities of optimal epitope-specific CD8+ T cells. Most importantly, the response to and protection against an in vivo CMV challenge were identical in adult and aged RMs. These data indicate that CMV-specific T cell immunity is well maintained in old RMs and argue against a primary role for progressive dysfunction of these responses in the development of immune senescence.

Polyinosinic-Polycytidylic Acid Is the Most Effective TLR Adjuvant for SIV Gag Protein–Induced T Cell Responses In Nonhuman Primates

Prime-boost immunization with heterologous vaccines elicits potent cellular immunity. In this study, we assessed the influence of various TLR ligands on SIV Gag–specific T cell immunity and protection following prime-boost immunization. Rhesus macaques (RMs) were primed with SIV Gag protein emulsified in Montanide ISA51 with or without TLR3 (polyinosinic-polycytidylic acid [poly-IC]), TLR4 (monophosphoryl lipid A), TLR7/8 (3M-012), TLR9 (CpG), or TLR3 (poly-IC) combined with TLR7/8 ligands, then boosted with replication defective adenovirus 5 expressing SIV Gag (rAd5-Gag). After priming, RMs that received SIV Gag protein plus poly-IC developed significantly higher frequencies of SIV Gag–specific CD4+ Th1 responses in blood and bronchoalveolar lavage (BAL) fluid lymphocytes compared with all other adjuvants, and low-level SIV Gag–specific CD8+ T cell responses. After the rAd5-Gag boost, the magnitude and breadth of SIV Gag–specific CD8+ T cell responses were significantly increased in RM primed with SIV Gag protein plus poly-IC, with or without the TLR7/8 ligand, or CpG. However, the anamnestic, SIV Gag–specific CD8+ T cell response to SIVmac251 challenge was not significantly enhanced by SIV Gag protein priming with any of the adjuvants. In contrast, the anamnestic SIV Gag–specific CD4+ T cell response in BAL was enhanced by SIV Gag protein priming with poly-IC or CpG, which correlated with partial control of early viral replication after SIVmac251 challenge. These results demonstrate that prime-boost vaccination with SIV Gag protein/poly-IC improves magnitude, breadth, and durability of CD4+ T cell immune responses, which could have a role in the control of SIV viral replication.

Naive T cells are dispensable for memory CD4+ T cell homeostasis in progressive simian immunodeficiency virus infection

Memory CD4+ T cell homeostasis and AIDS progression are independent of naive CD4+ T cells in SIV infection of nonhuman primates.

Optimization and qualification of an 8-color intracellular cytokine staining assay for quantifying T cell responses in rhesus macaques for pre-clinical vaccine studies.

Vaccination and SIV challenge of macaque species is the best animal model for evaluating candidate HIV vaccines in pre-clinical studies. As such, robust assays optimized for use in nonhuman primates are necessary for reliable ex vivo measurement of immune responses and identification of potential immune correlates of protection. We optimized and qualified an 8-color intracellular cytokine staining assay for the measurement of IFNγ, IL-2, and TNF from viable CD4 and CD8 T cells from cryopreserved rhesus macaque PBMC stimulated with peptides. After optimization, five laboratories tested assay performance using the same reagents and PBMC samples; similar results were obtained despite the use of flow cytometers with different configurations. The 8-color assay was then subjected to a pre-qualification study to quantify specificity and precision. These data were used to set positivity thresholds and to design the qualification protocol. Upon completion of the qualification study, the assay was shown to be highly reproducible with low inter-aliquot, inter-day, and inter-operator variability according to the qualification criteria with an overall variability of 20-40% for each outcome measurement. Thus, the 8-color ICS assay was formally qualified according to the ICH guidelines Q2 (R1) for specificity and precision indicating that it is considered a standardized/robust assay acceptable for use in pre-clinical trial immunogenicity testing.

Heterogeneity of CD4+ and CD8+ T-cell Responses to Cytomegalovirus in HIV-Infected and HIV-Uninfected Men Who Have Sex With Men

Studies of T-cell immunity to human cytomegalovirus (CMV) primarily reflect anti-CMV pp65 or immediate early antigen 1 (IE-1) activity. We assessed responses of T cells from human immunodeficiency virus (HIV)-negative and HIV-infected men to peptide pools spanning 19 CMV open reading frames selected because they previously correlated with total CMV-specific T-cell responses in healthy donors. Cells producing cytokines in response to pp65 or IE-1 together composed <12% and <40% of the total CD4(+) and CD8(+) T-cell responses to CMV, respectively. These proportions were generally similar regardless of HIV serostatus. Thus, analyses of total CMV-specific T-cell responses should extend beyond pp65 and IE-1 regardless of HIV serostatus.