UCLA

Although it is well-documented that T cells are crucial in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) yet the dynamic interplay between HIV-1 and T cells has not been fully elucidated. The effects that HIV-1 has on T cell diversity and the effects that T cell diversity has on HIV viral escape have not been well characterized; an understanding of these effects could have crucial implications for design of CTL vaccines. In particular, such information could provide insights needed to develop methods for reconstitution of sufficient diversity in the immune systems of HIV+ persons to allow CTL vaccines to be effective. Furthermore, such information could be helpful in the development of CTL vaccines against semi-conserved epitopes, so as to prevent viral escape. These are the aims this dissertation will attempt to address.

One major problem with the current approach to HIV-1 vaccine development is that the strategies currently being employed ultimately fail; this is mostly, but not entirely, due to HIV-1's high rate of mutation. This ultimately results in the escape of the virus from vaccine-induced immunity, thereby rendering such vaccines useless. Both CD4+ and CD8+ T cells play a major role in immune responses to HIV-1. However, during the course of infection, CD4+ T cells are depleted, not only in number, but also in diversity. Limited CD4+ T cell diversity cripples the immune system, as such CD4+ T cells are not able to provide the help necessary for effective innate and adaptive immune system responses to HIV, including help to CTL, which is of particular importance for this dissertation. CTL responses constitute one of the crucial arms of the immune system that is highly responsible for responding to HIV-1 infection. However, immune defenses mediated by CTL ultimately fail in HIV infection, which is, again, also largely (but not entirely) due to high rates of HIV-1 mutation that cause constant viral escape, which, in turn, drives chronic immune activation and ultimately CTL exhaustion.

We have addressed each of these problems in this dissertation. In Chapter Two, we present results of studies in which we examined thymic output and CD4+ T cell diversity from HIV+ persons who were perinatally infected, were in treatment and had lived with the infection for over two decades. In Chapter Three, we present results of studies in which we screened for CTL responses against the gag 162-173 KAFSPEVIPMF epitope from multiple persons and identified and cloned the TCR responsible for these responses, using a novel technique TCR identification and cloning technique that we also present in this chapter. Finally, we functionally tested the cloned KF11-specific TCR to confirm that this panel was able to recognize and lyse the most common circulating variants of the KF11 epitope.

The results presented in Chapter Two of this dissertation show that HIV+ participants had reduced CD4+ T cell levels, with predominant depletion of the memory subset, but preservation of naive cells. In most of these HIV+ participants, levels of CD4+ T cells that were recent thymic emigrants' CD4+ T cell levels were normal, and enhanced thymopoiesis was present, as indicated by higher proportions of CD4+ T cells containing TCR recombination excision circles. Memory CD4+ T cell depletion was highly associated with CD8+ T-cell activation in HIV-1-infected persons, and plasma interlekin-7 levels were correlated with levels of naive CD4+ T cells, suggesting activation-driven loss and compensatory enhancement of thymopoiesis. Deep sequencing of CD4+ T cell receptor sequences in HIV+ subjects who had high levels of compensatory enhancement of thymopoiesis revealed supranormal TCR diversity, providing additional evidence of enhanced thymic output.

In Chapter Three we introduce and describe an inexpensive new technique to quickly and efficiently identify, clone and functionally test epitope-specific TCR. Using this new technique and samples from multiple HIV+ HLA-B*5701 persons, we identified, cloned and functionally tested four KF11-specific TCR. The four identified KF11-specific TCR were able to recognize and lyse target cells that were peptide-loaded with the six most common circulating variants of KF11. These six variants make up 97% of all circulating variants, according to the Los Alamos HIV database. The functional avidity and killing efficiency of the KF11-specific TCR were also investigated. Consonant with prior supporting data on KF11-specific TCR, the functional avidity observed for these four KF11-specific TCR had a range of 89 ng/ml to 832 ng/ml. One of the KF11-specific TCR was tested for its ability to lyse HIV-infected cells. This TCR was able to lyse cells infected with three of the four variants that were previously recognized and lysed in the peptide-loaded target cells. If these TCR are validated in vivo, and they are to prevent viral escape, the process could be repeated with other HLA restricted epitopes in order to develop a new treatment against HIV-1.