Murine CD4+ T cell clones vary in function in vitro and in influenza infection in vivo

ABSTRACTThe expression of xenogeneic TRIM5α proteins can restrict infection in various retrovirus/host cell pairings. Previously, we have shown that African green monkey TRIM5α (AgmTRIM5α) potently restricts both human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus mac239 (SIVmac239) replication in a transformed human T-cell line (L. V. Coren, et al., Retrovirology 12:11, 2015,http://dx.doi.org/10.1186/s12977-015-0137-9). To assess AgmTRIM5α restriction in primary cells, we transduced AgmTRIM5α into primary rhesus macaque CD4 T cells and infected them with SIVmac239. Experiments with T-cell clones revealed that AgmTRIM5α could reproducibly restrict SIVmac239replication, and that this restriction synergizes with an intrinsic resistance to infection present in some CD4 T-cell clones. AgmTRIM5α transduction of virus-specific CD4 T-cell clones increased and prolonged their ability to suppress SIV spread in CD4 target cells. This increased antiviral function was strongly linked to decreased viral replication in the AgmTRIM5α-expressing effectors, consistent with restriction preventing the virus-induced cytopathogenicity that disables effector function. Taken together, our data show that AgmTRIM5α restriction, although not absolute, reduces SIV replication in primary rhesus CD4 T cells which, in turn, increases their antiviral function. These results support priorin vivodata indicating that the contribution of virus-specific CD4 T-cell effectors to viral control is limited due to infection.IMPORTANCEThe potential of effector CD4 T cells to immunologically modulate SIV/HIV infection likely is limited by their susceptibility to infection and subsequent inactivation or elimination. Here, we show that AgmTRIM5α expression inhibits SIV spread in primary effector CD4 T cellsin vitro. Importantly, protection of effector CD4 T cells by AgmTRIM5α markedly enhanced their antiviral function by delaying SIV infection, thereby extending their viability despite the presence of virus. Ourin vitrodata support priorin vivoHIV-1 studies suggesting that the antiviral CD4 effector response is impaired due to infection and subsequent cytopathogenicity. The ability of AgmTRIM5α expression to restrict SIV infection in primary rhesus effector CD4 T cells now opens an opportunity to use the SIV/rhesus macaque model to further elucidate the potential and scope of anti-AIDS virus effector CD4 T-cell function.

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Development of a Nonhuman Primate Model for Analysis of the Adoptive Transfer of Antigen-Specific T Cell Clones.

Blood ◽

10.1182/blood.v106.11.770.770 ◽

2005 ◽

Vol 106 (11) ◽

pp. 770-770

Author(s):

Carolina Berger ◽

Michael Jensen ◽

Stanley R. Riddell

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Adoptive Transfer ◽

Cell Transfer ◽

Primate Model ◽

T Cell Clones ◽

Cell Clones

Abstract In principle, the adoptive transfer of T cell clones specific for antigens expressed by pathogens or malignant cells could be therapeutically effective and allow precise control of the specificity, function, and magnitude of T cell immunity. However, the infusion of large numbers of cultured T cells or T cell clones in clinical trials has frequently failed to eradicate tumors or provide long-term control of infection. This may be due in part to the acquisition of an effector phenotype by the T cells during in vitro culture, which reduces their ability to survive in vivo and establish an immune response of sufficient magnitude for sustained efficacy. Several approaches including the administration of cytokines such as IL15, or lymphodepletion prior to cell transfer might promote the establishment of T cell memory after T cell transfer. To facilitate the rational development of clinical trials of T cell therapy, we have employed a nonhuman primate model of adoptive T cell transfer in which culture conditions and cell doses identical to those in human studies are utilized, and designed strategies to permit rigorous analysis of the persistence, function, phenotype, and migration of transferred cells. CD8+ CTL specific for macaque CMV were detected using an overlapping peptide panel and cytokine flow cytometry, isolated as individual T cell clones by limiting dilution, and propagated to large numbers in vitro. The T cell clones were transduced to express an intracellular truncated CD19 (ΔCD19) surface marker to allow tracking and functional assessment of T cells in vivo, and enriched by immunomagnetic selection to high purity (>98%) prior to transfer. The persistence of transferred ΔCD19+ T cells in the blood and their migration to the bone marrow and lymph nodes was determined by flow cytometry after staining with anti CD19, CD8, and CD3 antibodies. The infusion of ΔCD19+CD8+ CTL (3 x 108/kg) was safe and the cells remained detectable in vivo for >5 months. ΔCD19+CD8+ T cells were easily detected in the blood 1 day after transfer at a level of 2.7% of CD8+ T cells and gradually declined over 56 days to a stable population of 0.15–0.2% of CD8+ T cells. At the time of transfer the ΔCD19+CD8+ T cells had an effector phenotype (CD62L− CD127−), but gradually converted to a CD62L+CD127+ memory phenotype in vivo. The infused T cells were found at high levels in lymph node and bone marrow at day 14 after transfer (1.4% and 2.5%, respectively) and the cells at these sites were predominantly CD62L+. The ΔCD19+CD62L+ T cells lacked direct lytic function and expressed low levels of granzyme B, consistent with memory T cells. Sorting of these cells from post-transfer PBMC showed that in vitro activation restored lytic activity. The transferred ΔCD19+CD62L+ T cells in post-infusion PBMC produced IFNγ and TNFα comparable to endogenous CMV-specific CD8+ CTL. These results demonstrate that a subset (5–10%) of transferred CD8+ CTL clones can persist long-term as functional memory T cells. The macaque CD8+ T cell clones are responsive to IL15 in vitro and a safe regimen for administering IL15 to macaques that boosts endogenous T cells has been identified. Studies are now in progress to determine if IL15 can enhance the efficiency with which effector and memory CD8+ T cell responses can be augmented after adoptive transfer of T cell clones.

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Establishing T Cell Memory by Adoptive Transfer of T Cell Clones.

Blood ◽

10.1182/blood.v108.11.866.866 ◽

2006 ◽

Vol 108 (11) ◽

pp. 866-866

Author(s):

Carolina Berger ◽

Michael C. Jensen ◽

Stanley R. Riddell

Keyword(s):

T Cells ◽

T Cell ◽

Adoptive Transfer ◽

Annexin V ◽

T Cell Memory ◽

T Cell Clones ◽

Cell Clones

Abstract Adoptive transfer of T cells has been employed to reconstitute T cell immunity to viruses such as cytomegalovirus (CMV) in immunodeficient allogeneic stem cell transplant (SCT) patients and is being investigated to treat malignancies. In the allogeneic SCT setting, the T cells are derived from the donor and need to be isolated as clones or highly pure populations to avoid graft-versus-host disease. CD8+ T cells can be divided into defined subsets including CD62L− effector memory (TEM) and central memory T cells (TCM) expressing the CD62L lymph node homing molecule. Both TCM and TEM can give rise to cytolytic effector T cells (TE) after antigen stimulation and can be expanded in vitro for immunotherapy. However, the potential of T cells derived from either the TEM or TCM subset to persist in vivo has not been investigated. We used a macaque model to determine whether reconstitution of T cell memory to CMV by adoptive transfer of CD8+ T cell clones depended on their origin from either the CD62L+ TCM or CD62L− TEM subset. T cell clones were retrovirally transduced to express the macaque CD19 or CD20 surface marker to allow tracking of T cells in vivo. Clones derived from both TCM and TEM had similar avidity and proliferative capacity in vitro, and had a TE phenotype (CD62L−CCR7−CD28−CD127−, granzyme B+). TCM and TEM-derived T cell clones were transferred to macaques at doses of 3–6×108/kg and were both detected in the blood one day after transfer at 1.2–2.7% (low dose) to 20–25% (high dose) of CD8+ T cells. However, the frequency of TEM-derived T cells was undetectable after 3–5 days, and the cells were not present in lymph node or bone marrow obtained at day 14. By contrast, TCM-derived clones persisted in peripheral blood, migrated to tissue sites, and were detectable long-term at significant levels. A distinguishing feature of TCM-derived cells was their responsiveness to homeostatic cytokines. Only TCM-derived clones were rescued from apoptotic cell death by low-dose IL15 for >30 days in vitro and this correlated with higher levels of IL15Rα, IL2Rβ, and IL2Rγ, and of Bcl-xL and Bcl-2, which promote cell survival. To determine if the inability of TEM-derived clones to survive in vitro correlated with an increased susceptibility of cell death in vivo, we measured the proportion of infused cells that were positive for propidium iodide (PI) and Annexin V during the short period of in vivo persistence. One day after transfer, 41–45% of TEM-derived T cells were Annexin V+/PI+, analyzed directly in the blood or after 24 hours of culture. By contrast, only a minor fraction of an adoptively transferred TCM-derived T cell clone was Annexin V+/PI+ and the infused cells survived in vivo. A subset of the persisting T cells reacquired TCM marker (CD62L+CCR7+CD127+CD28+) in vivo and regained functional properties of TCM (direct lytic activity; rapid proliferation to antigen). These T cells produced IFN-γ and TNF-α after peptide stimulation, and studies are in progress to assess their in vivo response to antigen by delivery of T cells expressing CMV proteins. Our studies in a large animal model show for the first time that CD8+ TE derived from TCM but not TEM can persist long-term, occupy memory T cell niches, and restore TCM subsets of CMV-specific immunity. Thus, taking advantage of the genetic programming of cells that have become TCM might yield T cells with greater therapeutic activity and could be targeted for human studies of T cell therapy for both viral and malignant disease.

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