Direct cell-to-cell spread of human immunodeficiency virus type 1 (HIV-1) between T cells at the virological synapse (VS) is an efficient mechanism of viral dissemination. Tetherin (BST-2/CD317) is an interferoninduced, antiretroviral restriction factor that inhibits nascent cell-free particle release. The HIV-1 Vpu protein antagonizes tetherin activity; however, whether tetherin also restricts cell-cell spread is unclear. We performed quantitative cell-to-cell transfer analysis of wild-type (WT) or Vpu-defective HIV-1 in Jurkat and primary CD4 ؉ T cells, both of which express endogenous levels of tetherin. We found that Vpu-defective HIV-1 appeared to disseminate more efficiently by cell-to-cell contact between Jurkat cells under conditions where tetherin restricted cell-free virion release. In T cells infected with Vpu-defective HIV-1, tetherin was enriched at the VS, and VS formation was increased compared to the WT, correlating with an accumulation of virus envelope proteins on the cell surface. Increasing tetherin expression with type I interferon had only minor effects on cell-to-cell transmission. Furthermore, small interfering RNA (siRNA)-mediated depletion of tetherin decreased VS formation and cell-to-cell transmission of both Vpu-defective and WT HIV-1. Taken together, these data demonstrate that tetherin does not restrict VS-mediated T cell-to-T cell transfer of Vpu-defective HIV-1 and suggest that under some circumstances tetherin might promote cell-to-cell transfer, either by mediating the accumulation of virions on the cell surface or by regulating integrity of the VS. If so, inhibition of tetherin activity by Vpu may balance requirements for efficient cell-free virion production and cell-to-cell transfer of HIV-1 in the face of antiviral immune responses.

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Naturally occurring CD4 + CD25 hi Foxp3 + Tregs (nTregs) are highly proliferative in blood. However, the kinetics of their accumulation and proliferation during a localized antigen-specific T cell response is currently unknown. To explore this, we used a human experimental system whereby tuberculin purified protein derivative (PPD) was injected into the skin and the local T cell response analyzed over time. The numbers of both CD4 + Foxp3 -(memory) and CD4 + Foxp3 + (putative nTreg) T cells increased in parallel, with the 2 populations proliferating at the same relative rate. In contrast to CD4 + Foxp3 -T cell populations, skin CD4 + Foxp3 + T cells expressed typical Treg markers (i.e., they were CD25 hi , CD127 lo , CD27 + , and CD39 + ) and did not synthesize IL-2 or IFN-γ after restimulation in vitro, indicating that they were not recently activated effector cells. To determine whether CD4 + Foxp3 + T cells in skin could be induced from memory CD4 + T cells, we expanded skin-derived memory CD4 + T cells in vitro and anergized them. These cells expressed high levels of CD25 and Foxp3 and suppressed the proliferation of skin-derived responder T cells to PPD challenge. Our data therefore demonstrate that memory and CD4 + Treg populations are regulated in tandem during a secondary antigenic response. Furthermore, it is possible to isolate effector CD4 + T cell populations from inflamed tissues and manipulate them to generate Tregs with the potential to suppress inflammatory responses. IntroductionNaturally occurring CD4 + CD25 hi Foxp3 + Tregs (nTregs) can prevent reactivity to both self and non-self antigens (1-4). Although early studies suggested that these cells are generated as a distinct population in the thymus, CD4 + CD25 hi Foxp3 + Tregs, which are phenotypically and functionally identical to the thymus-derived population, can also be generated after antigen-induced proliferation of CD4 + T cells in peripheral tissues in mice (5, 6). The rapid division of CD4 + CD25 hi Foxp3 + Tregs that has been shown to occur in vivo in mice (7) and humans (8) may be a mechanism for maintaining nTregs. This has particularly important implications for the lifelong maintenance of human Tregs after thymic involution, since CD4 + CD25 hi Foxp3 + T cells in humans have limited capacity for extensive self-renewal, due to short telomeres, and lack telomerase activity (8). It is important to consider the possible difference in behavior and characteristics of nTregs in mice and humans, especially given the potential for species-specific differences that might lead to side effects during therapy (9).The regulation of immunity and pathology by intervention at the Treg axis has been very successful in animal models, where it has been shown that CD4 + CD25 hi Foxp3 + T cells can be harnessed to prevent autoimmunity (10, 11), inflammatory disease (12),

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