Adoptive transfer experiments may help elucidate the mechanism and limits of CD8+
T cell control of viral replication, which have important implications for vaccine design. In particular, it has been postulated that naïve and even vaccinated animals suffer from insufficient SIV-specific cells arriving at sites of viral replication (43
). To determine whether virus-specific CD8+
T cells can control SIV replication when infused in large numbers, we adoptively transferred 107
SIV-specific hemiallogeneic CD8+
T cells at or around the time of challenge in four rhesus macaques. While we did not observe any reduction in acute plasma viremia, the infused CD8+
T cells were generally undetectable in peripheral blood beyond 3 days. It is therefore difficult to conclude whether host rejection of the clones interfered with their antiviral activity or the clones were simply ineffective. To eliminate the prospect of rejection, we modified our protocol and infused autologous SIV-specific CD8+
T-cell clones to the same cohort of monkeys during chronic infection. While a markedly improved persistence was observed for the autologous cells, there was no measurable impact on the plasma viral load or CD4+
T-cell-associated SIV DNA. Although we were unable to detect any evidence of virus control by the transferred cells, tracking and phenotypic characterization of the infused cells in vivo
revealed some novel immunologic phenomena: a distinct preference for residing in the lungs and continuous expression of activation markers without apparent cell division. Together, these data suggest that an activated T cell phenotype may facilitate recruitment to specific effector/mucosal sites, such as the lungs, and does not necessarily reflect recent antigen stimulation.
The lack of perceptible effect on viral replication by either the hemiallogeneic or autologous CD8+ T cell adoptive transfer may be due to the inability of SIV-specific CD8+ T cells to independently control viremia, limitations of this adoptive transfer model, or a combination of these or related factors. The short persistence of the hemiallogeneic clones suggests that the host rejected them within days of the transfer, as autologous clones expanded and infused in the same manner persisted longer in our chronic infection model and during acute infection, as shown by Minang et al. (submitted for publication). This may explain the apparent accelerated rate of acute SIV replication observed in the two animals challenged the day after the transfer, as inflammation during a host-versus-graft response might increase the pool of activated CD4+ T cells susceptible to SIV infection at the time of challenge. Rapid clearance of the hemiallogeneic cells may also explain why we did not observe selective pressure exerted on targeted viral epitopes in circulating virus, while Minang et al. (submitted for publication) observed a very early escape mutation following transfer of autologous cells that did persist.
The question remains why there was no measurable effect of the autologous transfer on viremia. It is possible that the transfer procedure or a host suppressive response impaired the activity of the clones in vivo
. However, transferred cells recovered from the lungs one month following transfer in similar experiments performed during acute infection retained effector function upon ex vivo
antigen-specific stimulation (Minang et al
., submitted for publication). In addition, several clones expressed little or no PD-1 at the time of infusion. And although our infusions of clones originating from TCM
phenotypes did not show a difference in persistence, as the study by Berger et al.
), related studies also failed to detect any difference between TCM
- and TEM
-derived clones in terms of in vivo
persistence or anti-viral activity (Minang et al
., submitted for publication). Thus, the importance of memory phenotype in determining transferred cell fate is uncertain, and other barriers possibly limited their efficacy. Most likely, by retention in the lungs (or other tissues not sampled), the clones were sequestered from critical sites of virus replication. In addition, some (but not all) of the CD8+
T cell epitopes targeted by the autologous SIV-specific clones were found to have mutated prior to the infusion during chronic viremia, rendering those clones of limited value. It is also possible that soluble factors that suppress HIV-specific CD8+
T cell lytic activity during chronic HIV infection may impact SIV-specific cells as well (44
). Alternatively, inflammatory cytokines released by the effector cells may activate latently infected cells or promote new infection of recently activated CD4+
T cells, inadvertently boosting virus production. This could explain the transient spike in plasma viremia observed in the days post-infusion. Notably, autologous HIV-specific CD8+
T cell therapy in humans resulted in a similar elevation in plasma viremia (19
Although we could not detect an impact on viremia, the in vivo
trafficking of the infused clones revealed striking localization properties. When delivered i.v., autologous cells instantaneously populated peripheral blood, where they persisted for at least two months but declined steadily. These cells were highly abundant in the BAL, where they persisted to a greater extent than in the blood, dropping only 10-fold from peak representation compared to a 2000-fold reduction in the blood over 8 weeks. Distinct accumulation of the transferred cells in the lungs is consistent with entrapment of i.v. transferred effector cells as they pass through the constricting vasculature of this organ during pulmonary circulation. While similar observations have been made for adoptively transferred cells in mice (46
), to our knowledge this, in conjunction with similar findings by Minang et al
. (submitted for publication), is the first documentation of such distribution in primates. Homing to the lungs may be due to expression of adhesion molecules, such as lymphocyte function-associated antigen-1 (LFA-1), which mediate T cell entry into the airways (48
). There is also evidence that the morphological rigidity or polarization of effector cells contributes to trapping within pulmonary capillaries (51
). Adoptive transfer of bulk, unstimulated PBMC does not result in accumulation of the transferred cells in the lungs in cynomolgus macaques (53
), suggesting that this phenomenon may be dependent on the activated effector phenotype of the transferred cells. Notably, expression of α4β7 and CD103, markers commonly associated with intestinal homing, did not result in detectable localization of clones to the jejunum, as was also observed by Minang et al
. (submitted for publication).
The different localization and tissue equilibration patterns observed for i.p. and i.v. infused cells suggest that i.p. transfer largely avoids a massive, early lung entrapment. Rather, the slight, gradual increase in infused cells in BAL combined with the persistent low-level maintenance in blood may reflect slow leakage of peritoneal-deposited cells into circulation, followed by entrapment in the lungs as described above. This would provide a steady supply of i.p. delivered cells to continuously populate the BAL via the blood, which has been proposed to be a site of recruitment for memory T cells to the lungs (54
Infused cells that persisted in the lungs displayed an “activated” CD69+
expression profile; remarkably, a substantial fraction of these cells did not divide in vivo
for at least 6 weeks while maintaining this phenotype. Thus expression of either CD69 or HLA-DR, two markers commonly used to identify activated cells in PBMC or in ex vivo
analyses, does not necessarily indicate active cell division and should not be equated with concurrent effector functions. Moreover, this phenotype was typical of the resident lung CD8+
T cell population, suggesting that the lung contains a resting or semi-resting, long-lived, stable and nondividing T lymphocyte population. Similar evidence of nondividing T cells displaying an effector phenotype in the mouse lung has given rise to the theory that tissue-specific immunoregulatory mechanisms may exist to maintain homeostasis amidst high antigen load (55
). Indeed, this same phenotype for T cells has been seen for gut-associated CD4+
T cells (60
). Thus the hypothesis that the gut is a repository for highly-activated dividing T cells that serve as an optimal reservoir of SIV and HIV replication should be re-examined. In any case, the expression of “activation” markers on cells in vivo
cannot be used to definitively denote those cells as mitotically active nor even necessarily evincing effector functions.
In summary, we found no evidence of independent SIV control by infused CD8+ T cells. The capability of the transferred cells to target virus replication in vivo may have been hampered by several factors, including prompt host-versus-graft rejection for the hemiallogeneic clones infused during acute infection, homing to and confinement within peripheral tissues for many of the clones, viral epitope escape during chronic infection, or simply an insufficiency of cell number. Regardless, we demonstrated the feasibility of expanding massive numbers of antigen-specific CD8+ T-cell clones, while maintaining a memory phenotype and cytolytic activity. Moreover, infusion of over 1010 cells was well tolerated without any associated adverse events in the animals. We identified persistent transferred cells expressing activation markers that did not proliferate in vivo, suggesting that identification of effector and dividing cells by surface phenotype should be reconsidered. Our findings on T cell trafficking, proliferation, and survival following adoptive transfer will help to design future studies addressing CD8+ T-cell control of immunodeficiency virus replication in vivo.