Kendall A. Smith

Latent infection of CD4 + T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy

Combination therapy for HIV-1 infection can reduce plasma virus to undetectable levels, indicating that prolonged treatment might eradicate the infection. However, HIV-1 can persist in a latent form in resting CD4+ T cells. We measured the decay rate of this latent reservoir in 34 treated adults whose plasma virus levels were undetectable. The mean half-life of the latent reservoir was very long (43.9 months). If the latent reservoir consists of only 1 × 105 cells, eradication could take as long as 60 years. Thus, latent infection of resting CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective anti-retroviral therapy.

T‐Cell Growth Factor

The mRNA of the Gibbon ape T-Cell Growth Factor concentrated from a Cell line identified as UCD-MLA-144 lead to. the production and cloning of its cDNA and the elucidation of its gene structure and that of the mature protein. Transfer vectors for production of the Gibbon ape T-Cell Growth Factor are disclosed along with useful intermediate expression products.

Prolonged Interleukin-2Rα Expression on Virus-Specific CD8+ T Cells Favors Terminal-Effector Differentiation In Vivo

CD25, the high-affinity interleukin-2 (IL-2) receptor alpha chain, is rapidly upregulated by antigen-specific CD8(+) T cells after T cell receptor stimulation. Here, we demonstrate that during an acute viral infection, CD25 expression is quite dynamic-after initial upregulation, a subset of virus-specific T cells sustains CD25 expression longer than the rest. At this time when there is distinct heterogeneity in CD25 expression, examination of the in vivo fate of effector cells revealed that CD25(lo) cells, which are relatively less sensitive to IL-2, preferentially upregulate CD127 and CD62L and give rise to functional long-lived memory cells. In contrast, CD25(hi) cells perceiving prolonged IL-2 signals proliferate more rapidly, are prone to apoptosis, exhibit a more pronounced effector phenotype, and appear to be terminally differentiated. Consistent with this, sustained IL-2 receptor signaling during expansion drove terminal-effector differentiation. These data support the hypothesis that prolonged IL-2 signals during priming promote terminal-effector differentiation.

Therapeutic use of IL-2 to enhance antiviral T-cell responses in vivo.

Interleukin (IL)-2 is currently used to enhance T-cell immunity but can have both positive and negative effects on T cells. To determine whether these opposing results are due to IL-2 acting differently on T cells depending on their stage of differentiation, we examined the effects of IL-2 therapy during the expansion, contraction and memory phases of the T-cell response in lymphocytic choriomeningitis virus (LCMV)–infected mice. IL-2 treatment during the expansion phase was detrimental to the survival of rapidly dividing effector T cells. In contrast, IL-2 therapy was highly beneficial during the death phase, resulting in increased proliferation and survival of virus-specific T cells. IL-2 treatment also increased proliferation of resting memory T cells in mice that controlled the infection. Virus-specific T cells in chronically infected mice also responded to IL-2 resulting in decreased viral burden. Thus, timing of IL-2 administration and differentiation status of the T cell are critical parameters in designing IL-2 therapies.

Heterogeneity of human T-cell growth factor(s) due to variable glycosylation

Abstract T-cell growth factor (TCGF) prepared from human tonsil cells was shown to separate into multiple peaks of activity on isoelectric focusing gels and SDS-PAGE. Treatment with neuraminidase and glycosidases or inhibition of glycosylation, however, demonstrated that this heterogeneity was primarily due to sialylation and, to a lesser extent, differences in other sugar residues. In contrast, TCGF prepared from a human T-leukemia cell line was uniform in charge and size and indistinguishable from the asialo form of tonsil-derived activity. Both sialylated and asialo-TCGF mediated continuous in vitro T-cell proliferation over successive passages. Thus, the evidence is consistent with a variably glycosylated protein as the entity responsible for T-cell growth.

Interleukin‐2 Deficient Mice: A New Model to Study Autoimmunity and Self‐Tolerance

Previous work revealed IL-2 as a key cytokine regulating the growth, differentiation and function of lymphocytes (for review see Smith 1988. Paul 1989). It is a glycoprotein of about 15 kD (Gillis et al. 1978) which mediates cell communications via a complex receptor system composed of three subunits. apy (for review see Taniguchi & Minami 1993. Kishimoto et al. 1994). Only the a-chain is specific for IL-2. the p-subunit is used also by IL-15 (Giri et al. 1994. Burton et al. 1994) and the y-subunit is used by IL-2, IL-4. IL-7. lL-9 and IL-15 (Noguchi et al. 1993, Russell et al. 1993. Kondo et al. 1993). Since IL-2 is considered as an indispensable factor for cytotoxic responses, it is used as an immunotherapeutic agent for the treatment of patients with disseminated cancers and with infectious diseases (Rosenberg 1988, Kaplan et al. 1991, Fauci 1993). However, the exact role of IL-2 in vivo is far from being understood. Its elucidation was hampered by a lack of suitable experimental systems that would allow to evaluate the pleiotropic activities of this interleukin in the context of the whole immune system. The technology of targeted mutagenesis in mouse germline developed in the laboratories of Capecchi and Smithies (Thomas & Capecchi 1989,

Single-cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response.

Understanding how the immune system decides between tolerance and activation by antigens requires addressing cytokine regulation as a highly dynamic process. We quantified the dynamics of interleukin‐2 (IL‐2) signaling in a population of T cells during an immune response by combining in silico modeling and single‐cell measurements in vitro. We demonstrate that IL‐2 receptor expression levels vary widely among T cells creating a large variability in the ability of the individual cells to consume, produce and participate in IL‐2 signaling within the population. Our model reveals that at the population level, these heterogeneous cells are engaged in a tug‐of‐war for IL‐2 between regulatory (Treg) and effector (Teff) T cells, whereby access to IL‐2 can either increase the survival of Teff cells or the suppressive capacity of Treg cells. This tug‐of‐war is the mechanism enforcing, at the systems level, a core function of Treg cells, namely the specific suppression of survival signals for weakly activated Teff cells but not for strongly activated cells. Our integrated model yields quantitative, experimentally validated predictions for the manipulation of Treg suppression.

CR6 : A third member in the MyD118 and Gadd45 gene family which functions in negative growth control

MyD118 and Gadd45 are two related genes which encode for proteins that play important roles in negative growth control, including both growth suppression and apoptosis. A strategy was employed to clone new members of the MyD118 and Gadd45 family of genes. Based on alignment of the deduced amino acid sequences, one cDNA clone was found to encode for the murine homologue of human CR6, originally cloned as an IL-2 immediate-early response gene. The murine and human CR6 proteins were observed to be 97% identical, indicating that CR6 is an evolutionarily conserved protein. Analysis of CR6 expression during hematopoietic cell development associated with growth arrest and apoptotic cell death, upon exposure of hematopoietic cells to a variety of growth arrest and apoptotic stimuli, and in a variety of murine tissues, has revealed that CR6 expression differs significantly from the expression of the related MyD118 and Gadd45 genes. Nevertheless, CR6, like MyD118 and Gadd45, suppressed colony formation of human lung carcinoma H1299 cells. These data suggest that CR6 plays similar, but not identical, roles to MyD118 and Gadd45 in negative control of cell growth.

PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells

The inhibitory receptor programmed cell death 1 (PD-1) plays a major role in functional exhaustion of T cells during chronic infections and cancer, and recent clinical data suggest that blockade of the PD-1 pathway is an effective immunotherapy in treating certain cancers. Thus, it is important to define combinatorial approaches that increase the efficacy of PD-1 blockade. To address this issue, we examined the effect of IL-2 and PD-1 ligand 1 (PD-L1) blockade in the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We found that low-dose IL-2 administration alone enhanced CD8+ T cell responses in chronically infected mice. IL-2 treatment also decreased inhibitory receptor levels on virus-specific CD8+ T cells and increased expression of CD127 and CD44, resulting in a phenotype resembling that of memory T cells. Surprisingly, IL-2 therapy had only a minimal effect on reducing viral load. However, combining IL-2 treatment with blockade of the PD-1 inhibitory pathway had striking synergistic effects in enhancing virus-specific CD8+ T cell responses and decreasing viral load. Interestingly, this reduction in viral load occurred despite increased numbers of Tregs. These results suggest that combined IL-2 therapy and PD-L1 blockade merits consideration as a regimen for treating human chronic infections and cancer.

Vaccination of Macaques with Long-Standing SIVmac251 Infection Lowers the Viral Set Point After Cessation of Antiretroviral Therapy

A cohort of rhesus macaques with long-standing SIVmac251 infection (≥5 mo) was treated with continuous antiretroviral therapy (ART). A group of eight macaques was vaccinated with or without simultaneous administration of low dose IL-2 with the highly attenuated poxvirus vector (NYVAC) vaccine candidate expressing the SIVmac structural gag-pol-env (gpe) genes and a novel chimeric fusion protein derived from the rev-tat-nef (rtn) regulatory genes. Control groups consisted of mock-vaccinated macaques or animals treated only with IL-2. Vaccination significantly expanded both virus-specific CD4+ and CD8+ T cell responses, and IL-2 further increased the vaccine-induced response to an immunodominant Gag epitope. Following antiretroviral treatment interruption, the viral set point was significantly lower in vaccinated than in control macaques for at least 4 consecutive mo, and viral containment was inversely correlated with vaccine-induced, virus-specific CD4+ and CD8+ T cell responses. These data provide the proof of concept that therapeutic vaccination before cessation of ART may be a feasible approach in the clinical management of HIV-1 infection.