Faculty Bibliography

Background: Increased tumor infiltrating lymphocytes (TILs) in primary (P) and locoregional melanoma tissue correlate with improved clinical outcome. Our recent data have suggested that matrix metalloproteinase 23 (MMP 23) expression (exp) in P result in lower prevalence of TILs and correlate with poor clinical outcome. On this basis, we examined P and metastatic (M) melanoma tissues to assess for concordance between the presence of TILs, MMP 23 protein levels and clinical response(resp) to anti-cytotoxic T-lymphocyte antigen 4 (CTLA4) therapy (tx). Methods: 21 melanoma patients (pts) with M specimens were analyzed. 17 matched P specimens were also evaluated. Immunohistochemical (IHC) staining for TILs of the pre-anti-CTLA4 specimens were conducted and confirmed by 2 pathologists. IHC TILs were graded- 2+: >10% TILs present in multiple foci in both peri- and through the tumor; 1+: 1-10% TILs present in one or more foci in the tumor and predominantly peri-tumor; 0: no TILs were present or if the lymphocytes did not infiltrate the tumor. TILs in P and M were analyzed for concordance and potential for predictability of resp to anti-CTLA4 tx. Staining to identify lymphocyte subtypes and MMP 23 exp in M is being completed. Results: 20 pts received anti-CTLA4 tx. M analysis- 6 pts with 0 TILs in M (5 no response [NR], 1 partial response[PR]); 8 pts with 1-2+ TILs in M (1 complete response [CR], 5 PR, 2 progressive disease [PD]); 6 pts with 2+ TILs in M ( 3 CR, 2 PR, 1PD). 1 pt with 2+ TILs in M resected, no tx and 4 years disease free. TILs present in 13 P, absent in 4 P and not evaluable in 4 pts with unknown P melanoma. MMP 23 protein scores in P (range 2-4) correlated with melanoma recurrence. MMP 23 exp in M will be reported. Conclusions: TILs in P do not appear to correlate with TILs in M or predict for resp to anti-CTLA4 tx. TILs in M may be an indicator of responsiveness to anti-CTLA4 tx. Identification of the type of M TIL subsets may further refine tx recommendations

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs). This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity). It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment. Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV

alphabeta T cell receptor (TCR) recognition of foreign peptides bound to major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells is a key event in the initiation of adaptive cellular immunity. In vitro, high-affinity binding and/or long-lived interactions between TCRs and pMHC correlate with high-potency T cell activation. However, less is known about the influence of TCR/pMHC interaction parameters on T cell responses in vivo. We studied the influence of TCR/pMHC binding characteristics on in vivo T cell immunity by tracking CD4(+) T cell activation, effector, and memory responses to immunization with peptides exhibiting a range of TCR/pMHC half-lives and in vitro T cell activation potencies. Contrary to predictions from in vitro studies, we found that optimal in vivo T cell responses occur to ligands with intermediate TCR/pMHC half-lives. The diminished in vivo responses we observed to the ligand exhibiting the longest TCR/pMHC half-life were associated with attenuation of intracellular signaling, expansion, and function over a broad range of time points. Our results reveal a level of control over T cell activation in vivo not recapitulated in in vitro assays and highlight the importance of considering in vivo efficacy of TCR ligands as part of vaccine design

T cell selection and maturation in the thymus depends on the interactions between T cell receptors (TCRs) and different self-peptide-major histocompatibility complex (pMHC) molecules. We show that the affinity of the OT-I TCR for its endogenous positively selecting ligands, Catnb-H-2K(b) and Cappa1-H-2K(b), is significantly lower than for previously reported positively selecting altered peptide ligands. To understand how these extremely weak endogenous ligands produce signals in maturing thymocytes, we generated soluble monomeric and dimeric peptide-H-2K(b) ligands. Soluble monomeric ovalbumin (OVA)-K(b) molecules elicited no detectable signaling in OT-I thymocytes, whereas heterodimers of OVA-K(b) paired with positively selecting or nonselecting endogenous peptides, but not an engineered null peptide, induced deletion. In contrast, dimer-induced positive selection was much more sensitive to the identity of the partner peptide. Catnb-K(b)-Catnb-K(b) homodimers, but not heterodimers of Catnb-K(b) paired with a nonselecting peptide-K(b), induced positive selection, even though both ligands bind the OT-I TCR with detectable affinity. Thus, both positive and negative selection can be driven by dimeric but not monomeric ligands. In addition, positive selection has much more stringent requirements for the partner self-pMHC

Rapid binding of peptides to MHC class II molecules is normally limited to a deep endosomal compartment where the coordinate action of low pH and HLA-DM displaces the invariant chain remnant CLIP or other peptides from the binding site. Exogenously added peptides are subject to proteolytic degradation for extended periods of time before they reach the relevant endosomal compartment, which limits the efficacy of peptide-based vaccines and therapeutics. In this study, we describe a family of small molecules that substantially accelerate the rate of peptide binding to HLA-DR molecules in the absence of HLA-DM. A structure-activity relationship study resulted in analogs with significantly higher potency and also defined key structural features required for activity. These compounds are active over a broad pH range and thus enable efficient peptide loading at the cell surface. The small molecules not only enhance peptide presentation by APC in vitro, but are also active in vivo where they substantially increase the fraction of APC on which displayed peptide is detectable. We propose that the small molecule quickly reaches draining lymph nodes along with the coadministered peptide and induces rapid loading of peptide before it is destroyed by proteases. Such compounds may be useful for enhancing the efficacy of peptide-based vaccines and other therapeutics that require binding to MHC class II molecules.

Several challenges face the development and operation of a biospecimen bank linked to clinical information, a critical component of any effective translational research program. Melanoma adds particular complexity and difficulty to such an endeavor considering the unique characteristics of this malignancy. We describe here a review of biospecimen bank and our experience in establishing a multi-disciplinary, prospective, integrated clinicopathological-biospecimen database in melanoma. The Interdisciplinary Melanoma Cooperative Group (IMCG), a prospective clinicopathological and biospecimen database, was established at the New York University (NYU) Langone Medical Center. With patients' informed consent, biospecimens from within and outside NYU, clinicopathological data, and follow-up information are collected using developed protocols. Information pertaining to biospecimens is recorded in 35 fields, and clinicopathological information is recorded in 371 fields within 5 modules in a virtual network system. Investigators conducting research utilizing the IMCG biospecimen resource are blind to clinicopathological information, and molecular data generated using biospecimens are linked independently with clinicopathological data by biostatistics investigators. This translational research enterprise acts as a valuable resource to efficiently translate laboratory discoveries to the clinic

The mechanisms by which alphabeta T-cells are selected in the thymus and then recognize peptide MHC (pMHC) complexes in the periphery remain an enigma. Recent work particularly with respect to quantification of T-cell sensitivity and the role of self-ligands in T-cell activation has provided some important clues to the details of how TCR signaling might be initiated. Here, we highlight recent experimental data that provides insights into the initiation of T-cell activation and also discuss the main controversies and uncertainties in this area.

In light of recent data showing that both helper and cytotoxic T cells can detect even a single molecule of an agonist peptide-MHC, alphabeta T cells are clearly a type of sensory cell, comparable to any in the nervous system. In addition, endogenous (self) peptides bound to MHCs are not just important for thymic selection, but also play an integral role in T cell activation in the response to foreign antigens. With the multitude of specificities available to most T cells, they can thus be considered as a sensory organ, trained on self-peptide-MHCs and primed to detect nonself