CD8 T cells are a critical component of the immune response against intracellular pathogens. In HIV infection, CD8 T cells help to contain viral replication after infection but do not completely clear infected cells. It is possible that if these CD8 T cell responses were logged into immunological memory after vaccination and present at the time of infection, they could prevent transmission, prevent the rapid dissemination of virus to tissue reservoirs or permanently control virus replication. Thus far, we have not defined the required quality, quantity, or focus of a CD8 T cell response that controls viral replication. As an important side-note, it is also unclear whether the responses that control viral replication after infection are the same responses that prevent HIV acquisition.
We used groups of completely MHC-matched MCM to study the CD8 T cell response while minimizing the confounding effects of host genetic diversity. We used eighteen animals with defined MHC haplotypes and performed 324 different E:T combinations. Specifically, we included animals that were M1/M1 homozygous, M3/M3 homozygous, and M1/M3 heterozygous to explore how MHC class I genes on one haplotype can affect the CD8 T cell responses restricted by the other haplotype. We also used this model to explore the breadth of the CD8 T cell response in heterozygous animals. Unlike tetramer stains that strictly examine the frequency of SIV-specific CD8 T cells or assays that use indirect assessments of CD8 T cell function through measuring cytokine production, viral suppression assays directly measure the ability of CD8 T cells to prevent virus replication. Using this assay, we were able to examine whether CD8 T cells from heterozygous animals suppress viral replication on target cells bearing zero, one or two matched MHC class I haplotypes. Better suppression on multiple haplotypes may indicate that animals have greater breadth of CD8 T cells that suppress viral replication or greater functional breadth.
We expected M1/M3 effector cells to recognize a greater variety of epitopes on M1/M3 target cells than on M1/M1 or M3/M3 target cells leading to greater suppression of viral replication on M1/M3 target cells. However, M1/M3 effector cells did not suppress viral replication most effectively on M1/M3 target cells. These results indicate that heterozygous animals do not mount a CD8 T cell response of greater functional breadth than homozygous animals. Moreover, we found that M1/M3 effector cells frequently suppressed viral replication most effectively on M1/M1 target cells suggesting that suppressive responses in these heterozygous animals were primarily directed against epitopes restricted by alleles on the M1 haplotype and that the contribution of the M3 responses is small. One explanation for these results is that M1/M1 animals are presenting a greater amount of M1-restricted peptides on the surface of their cells in comparison to M1/M3 target cells, facilitating recognition by CD8 T cells restricted by alleles encoded on the M1 haplotype.
The differences we observed in effector cell ability to suppress viral replication on M1/M3 target cells compared to matched target cells is particularly interesting. M3/M3 animals suppress viral replication significantly better on matched target cells than they do on heterozygous target cells. This is different from the M1/M1 animals that suppress viral replication well on MHC matched and heterozygous target cells. Combined, these findings indicate that there may be reduced presentation of M3 restricted epitopes on the surface of M1/M3 cells. These results mirror those from studies on other pathogens including HIV, CMV and EBV 
. Immunodominance is a multifaceted phenomenon and there are likely several mechanisms responsible for our observations. These may include the availability of antigenic peptides, the expression of different MHC class I alleles and the precursor frequencies of SIV-specific CD8 T cells 
. Ultimately, these results suggest that heterozygous animals will mount functional CD8 T cell responses of limited focus despite the potential capacity for infected cells to present a greater variety of peptides on their surface.
The apparent dominance of M1-restricted CD8 T cell responses in M1/M3 animals and M1/M1 animals, in addition to the observed correlation between the M1 haplotype and control of viral loads, suggests that these responses may have a protective role during SIV infection. While we were unable to correlate viral inhibition with viral loads, this may, in part, be due to the outgrowth of autologous virus in our assay. Nevertheless, the lower viral loads in M1+ animals indicate that CD8 T cells in these animals could be playing an essential role in the containment of viral replication 
. Our results may also have important implications for vaccine studies in other models like the rhesus or pigtail macaques which contain known ‘protective’ MHC class I alleles 
. Critically, specific responses restricted by these alleles may be responsible for control and vaccines targeted at generating responses of greater breadth may be better served by including minimal peptide antigens over whole protein constructs or focusing specifically on individual responses at the expense of epitopic breadth. At first glance, these findings appear negative for those with ‘non-protective’ MHC class I alleles, but it may be that protective responses in these individuals are subdominant. Vaccination for these subdominant responses may help non-controllers more effectively inhibit viral replication. Additionally, it is possible that, while our study does not indicate breadth is important to viral control, other models may provide different information or more nuanced views of the role of CD8 T cells in viral control.
Several additional factors might have played a role in the variable suppression we witnessed in this assay. First, the virus growing in target cells from animals with high viral loads may have contained CD8 T cell escape mutations. In a separate, but related study, we found that virus in M3/M3 animals escaped the HW8 CD8 T cell response which is correlated with in vivo
control of virus replication. The virus escaped with slower kinetics in animals that control viral loads like the M1 animals. This may have lead to a reduced capacity to suppress viral replication by M1/M1 effector cells. We do not have sequence data from the assay to assess whether the frequency of sequence variants correlated with suppression. Additionally, it may be possible that CD4 or CD8 T cell dysfunction played a role in reduced suppression. Vaccinated macaques may exhibit a different capacity to suppress viral replication on targets containing different MHC-haplotypes. Finally, it is not clear whether the suppression we observed is entirely SIV-specific or if the CD8 cell antiviral factor (CAF) is playing a role in the suppression we are observing in this assay 
These experiments would have been impossible in humans or other outbred macaque species. These studies represent the first time that large groups of MHC-matched animals could be studied in the context of AIDS-virus infection. Using groups of six MHC-matched animals made it possible to perform studies ex vivo that reached statistical significance and combined for 324 different E:T combinations. They provide a provocative finding that, if true, demonstrates some haplotypes contain certain alleles that are dominant to others in SIV infection. Nevertheless, we were unable to perform these studies in animals containing alternative MHC-haplotypes. It is possible that this finding is unique to these two MHC-haplotypes and that if one performed this experiment with HIV infected individuals, heterozygote effector cells might suppress equally well on targets bearing either matched MHC-haplotype. VSAs with MCM provide a unique model for understanding the functional breadth and efficacy of CD8 T cells in the context of SIV infection. By using this assay with other viruses or vaccine strategies, the VSA in MCM may prove useful to evaluating future HIV vaccine candidates.