Faculty Bibliography

The peptide segment of the second variable loop of HIV-1 spanning positions 166-181 harbors two functionally important sites. The first, spanning positions 179-181, engages the human alpha4beta7 integrin receptor which is involved in T-cell gut-homing and may play a role in human immunodeficiency virus (HIV)-host cell interactions. The second, at positions 166-178, is a major target of anti-V2 antibodies elicited by the ALVAC/AIDSVAX vaccine used in the RV144 clinical trial. Notably, these two sites are directly adjacent, but do not overlap. Here, we report the identity of a second determinant of alpha4beta7 binding located at positions 170-172 of the V2 loop. This segment - tripeptide QRV170-172- is located within the second site, yet functionally affects the first site. The absence of this segment abrogates alpha4beta7 binding in peptides bearing the same sequence from position 173-185 as the V2 loops of the RV144 vaccines. However, peptides exhibiting V2 loop sequences from heterologous HIV-1 strains that include this QRV170-172 motif bind the alpha4beta7 receptor on cells. Therefore, the peptide segment at positions 166-178 of the V2 loop of HIV-1 viruses appears to harbor a cryptic determinant of alpha4beta7 binding. Prior studies show that the anti-V2 antibody response elicited by the RV144 vaccine, along with immune pressure inferred from a sieve analysis, is directed to this same region of the V2 loop. Accordingly, the anti-V2 antibodies that apparently reduced the risk of infection in the RV144 trial may have functioned by blocking alpha4beta7-mediated HIV-host cell interactions via this cryptic determinant.

The immune-correlate analysis of the RV144 clinical trial revealed that human plasma IgA immune responses elicited by the RV144 vaccine correlated positively with a risk for HIV acquisition. This result once again emphasized that HIV vaccines can potentially have adverse effects leading to enhancement of infection. Here, we discuss previously reported evidence of antibody-dependent enhancement of HIV infection. We also describe how a structure-based epitope-specific sieve-analysis can be employed to mine the molecular mechanism underlying this phenomenon.

Adoptive immunotherapy with antigen-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor antigens are nonreactive "self" proteins, which presents an immunotherapy design challenge. Studies have shown that tumor-specific T cell receptors (TCRs) can be transduced into normal peripheral blood lymphocytes, which persist after transfer in about 30% of patients and effectively destroy tumor cells. Still, recent clinical trial with affinity-enhanced TCRs has resulted in severe effects due to cross reactivity to an unrelated peptide. Thus, the challenge for targeted T cell therapy remains to increase T cell potency in order to improve clinical responses and ensure on-target specificity by avoiding unwanted cross reactivity. We used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor differentiation antigen-major histocompatibility complex (pMHC), Mart-1(27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. Structural based approaches have been used to increase TCR affinity, however their potential cross-reactivity has not been reported. Here, we analyzed the effects of selected TCR point mutations alone and in combination on T cell activation potency. Further, we analyzed their specificity and cross-reactivity with related antigens presented by different melanoma cell lines and donor-derived antigen presenting cells. Our structure-based approach allowed us to rationally design sequence substitutions that improve binding in contact areas between the TCR and pMHC without increasing cross-reactivity with a wide variety of self-antigens. We identified and evaluated point mutations in critical TCR positions resulting in more potent T cell activation but maintaining overall specificity. When double and triple combination mutations were introduced, they exhibited an additive enhancement that further improved T cell activation while retaining a high degree of specificity. Conclusions: Such affinity-optimized TCRs could potentially be used in adoptive immunotherapy to treat melanoma while minimizing adverse autoimmunity effects

T cells play a critical role in host defense against viruses, intra- and extracellular microbes, and tumors. Because foreign antigen is presented amongst a vast majority of self-antigens, T cells have evolved the unique ability to discriminate "self" from "non-self" with high sensitivity and selectivity, enabling the elimination of foreign pathogens while largely avoiding self-reactivity. However, tissue-specific autoimmunity and tolerance to or eradication of cancer does not fit neatly into the self/non-self paradigm because the T cell responses in these situations are not directed to an exogenous pathogen, but rather most often to non-mutated self-proteins. Therefore, an important question is how the immune system establishes suitable thresholds that allow positively selected T cells to interact with selfligands in the periphery without causing overt activation. One hypothesis to explain how a T cell distinguishes among different types of self-ligands is the kinetic proof-reading theory, which relates signaling efficacy to the lifetime of the TCR (T cell receptor)-pMHC (peptide-major histocompatibility complex) interaction. More recently, T cell maturation associated signaling feedback pathways have also been hypothesized to play a role in T cell discrimination of between self-ligands. We are taking a variety of biophysical and cellular imaging approaches to determine how specific thresholds for T cell recognition of self-antigens are set. Our recent results [1] indicate that antitumor activity and autoimmunity are coupled and have a similar kinetic threshold; reducing autoimmunity cannot be accomplished without sacrificing efficacy of tumor killing. Therefore, an "optimal TCR affinity range" that leads to optimal tumor regression and minimal autoimmunity is elusive and treatment strategies focusing on increasing TCR affinities to a supraphysiological level has most likely little therapeutic benefit. Therefore, other approaches are needed to improve the balance between anti-tumor responses and autoimmunity. Our strategy to overcome this issue includes novel methods for careful biophysical engineering of tumor-specific TCRs to carefully balance tumorreactivity and autoimmunity. Furthermore, our recent preliminary data show that TCR-proximal signaling differs significantly between effector memory and central memory T cells due to differential constitutive activity and localization of signaling molecules. Understanding how activation signaling contributes to differences in memory T cell subset sensitivity may provide insight into how T cells can be manipulated to achieve optimal anti-tumor sensitivity. This could lead to adjuvants that target and enhance antigenspecific T cell anti-tumor efficacy. Together may lead to development of cancer immunotherapy approaches with improved outcomes