CD8 + T cell responses to viral infection are characterized by the emergence of dominant and subdominant CTL populations. The immunodominance hierarchies of these populations are highly reproducible for any given spectrum of virus-induced peptide-MHCI complexes and are likely determined by multiple factors. Recent studies demonstrate a direct correlation between naive epitope-specific CD8 + T cell precursor (CTLp) frequency and the magnitude of the response after antigen challenge. Thus, the number of available precursors in the naive pool has emerged as a key predictor of immunodominance. In contrast to this, we report here no consistent relationship between CTLp frequency and the subsequent magnitude of the immune response for 4 influenza virus-derived epitopes following intranasal infection of mice with influenza A virus. Rather, the characteristic, antigen-driven T cell immunodominance hierarchy was determined by the extent of recruitment from the available pool of epitope-specific precursors and the duration of their continued expansion over the course of the infection. These findings suggest possibilities for enhancing protective immune memory by maximizing both the size and diversity of typically subdominant T cell responses through rational vaccine design.

Characterizing the TCRα and TCRβ chains expressed by T cells responding to a given pathogen or underlying autoimmunity helps in the development of vaccines and immunotherapies, respectively. However, our understanding of complementary TCRα and TCRβ chain utilization is very limited for pathogen-and autoantigen-induced immunity. To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCRα and TCRβ chains from single cells. This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCRα chains and the need for prior knowledge of variable region usage in the TCRβ chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCRα and TCRβ chains with single-cell resolution. Using CD8 + CTLs specific for an influenza epitope recovered directly from the pneumonic lungs of mice, this technique determined that 25% of such effectors expressed a dominant, nonproductively rearranged Tcra transcript. T cells with these out-of-frame Tcra mRNAs also expressed an alternate, in-frame Tcra, whereas approximately 10% of T cells had 2 productive Tcra transcripts. The proportion of cells with biallelic transcription increased over the course of a response, a finding that has implications for immune memory and autoimmunity. This technique may have broad applications in mouse models of human disease. IntroductionRecent advances have allowed us to analyze the development and persistence of virus-specific CD8 + T cell-mediated immunity from naive CTL precursors (CTLps) in peripheral lymphoid tissue, through the antigen-driven phase in lymph nodes and spleen, to the CTL effectors in a site of virus-induced pathology, and then, ultimately, to the persistence and recall of immune memory (1-3). However, unless we use lymphocytes from TCR-transgenic mice, our capacity to follow the fate and persistence of defined clonotypes is very limited. Several approaches have been used to estimate the extent of TCR diversity and to track clonally expanded T cell populations throughout the course of antigen-specific CTL responses (4), but none has given the complete picture. A commonly used protocol is to double-stain CD8 + T cells with mAbs specific for TCR variable (V) region β (TRBV) and tetramers specific for peptide + class I MHC glycoprotein (pMHCI) epitopes (5-9). Such low-resolution analysis provides no insight into the extent of clonal diversity within a particular TRBV-specific population and offers little scope for determining the spectrum of TCRα usage, as there are few mAb reagents. Another approach, known as immunoscope or spectratyping, uses gel electrophoresis of total mRNA from TRBV-specific populations (RT-PCR product) to determine profiles of complementarity-determining region 3β (CDR3β) length (4, 10). Combining spectratyping with cloning and sequencing allows for more definitive identification of CDR3β transcripts, but the approach is compromised by the possibility of bias durin...

Breast cancer brain metastases are a deadly sequela of primary breast tumors that overexpress human epidermal growth factor receptor 2 (HER2); median survival for patients with these tumors is 10 to 13 months from the time of diagnosis. Current treatments for HER2-positive breast cancer brain metastases are invasive, toxic, and largely ineffective. Here, we have developed an adeno-associated virus serotype 9 (AAV9) vector to express the anti-HER2 monoclonal antibody trastuzumab (Herceptin) A single prophylactic intrathecal administration of AAV9.trastuzumab vector in a novel orthotopic murine xenograft model of HER2-positive breast cancer brain metastases significantly increased median survival, attenuated brain tumor growth, and preserved both the HER2 antigen specificity and the natural killer cell-associated mechanism of action of trastuzumab. When administered as a tumor treatment, AAV9.trastuzumab increased median survival. Dose-escalation studies revealed that higher doses of AAV9.trastuzumab resulted in smaller tumor volumes. Our results indicate that intrathecal AAV9.trastuzumab may provide significant antitumor activity in patients with HER2-positive breast cancer brain metastases. Intrathecal delivery of trastuzumab via adeno-associated virus has the potential to become a novel, integral part of adjuvant therapy for patients with HER2-positive breast cancer brain metastases. .

No abstract

<div>Abstract<p>Breast cancer brain metastases are a deadly sequela of primary breast tumors that overexpress human epidermal growth factor receptor 2 (HER2); median survival for patients with these tumors is 10 to 13 months from the time of diagnosis. Current treatments for HER2-positive breast cancer brain metastases are invasive, toxic, and largely ineffective. Here, we have developed an adeno-associated virus serotype 9 (AAV9) vector to express the anti-HER2 monoclonal antibody trastuzumab (Herceptin) <i>in vivo</i>. A single prophylactic intrathecal administration of AAV9.trastuzumab vector in a novel orthotopic <i>Rag1^−/−</i> murine xenograft model of HER2-positive breast cancer brain metastases significantly increased median survival, attenuated brain tumor growth, and preserved both the HER2 antigen specificity and the natural killer cell–associated mechanism of action of trastuzumab. When administered as a tumor treatment, AAV9.trastuzumab increased median survival. Dose-escalation studies revealed that higher doses of AAV9.trastuzumab resulted in smaller tumor volumes. Our results indicate that intrathecal AAV9.trastuzumab may provide significant antitumor activity in patients with HER2-positive breast cancer brain metastases.</p><p><b>Significance:</b> Intrathecal delivery of trastuzumab via adeno-associated virus has the potential to become a novel, integral part of adjuvant therapy for patients with HER2-positive breast cancer brain metastases. <i>Cancer Res; 78(21); 6171–82. ©2018 AACR</i>.</p></div>