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With age, the immune system becomes less effective, causing increased susceptibility to infection. Chronic CMV infection further impairs immune function and is associated with increased mortality in the elderly. CMV exposure elicits massive CD8 + T cell clonal expansions and diminishes the cytotoxic T cell response to subsequent infections, leading to the hypothesis that to maintain homeostasis, T cell clones are expelled from the repertoire, reducing T cell repertoire diversity and diminishing the ability to combat new infections. However, in humans, the impact of CMV infection on the structure and diversity of the underlying T cell repertoire remains uncharacterized. Using TCR β-chain immunosequencing, we observed that the proportion of the peripheral blood T cell repertoire composed of the most numerous 0.1% of clones is larger in the CMV seropositive and gradually increases with age. We found that the T cell repertoire in the elderly grows to accommodate CMV-driven clonal expansions while preserving its underlying diversity and clonal structure. Our observations suggest that the maintenance of large CMV-reactive T cell clones throughout life does not compromise the underlying repertoire. Alternatively, we propose that the diminished immunity in elderly individuals with CMV is due to alterations in cellular function rather than a reduction in CD8 + T cell repertoire diversity., (Copyright © 2019 The Authors.)

Immunotherapy with T cells genetically modified to express chimeric Ag receptors (CARs) that target tumor-associated molecules have impressive efficacy in hematological malignancies. The field has now embraced the challenge of applying this approach to treat common epithelial malignancies, which make up the majority of cancer cases but evade immunologic attack by a variety of subversive mechanisms. In this study, we review the principles that have guided CAR T cell design and the extraordinary clinical results being achieved in B cell malignancies targeting CD19 with a single infusion of engineered T cells. This success has raised expectations that CAR T cells can be applied to solid tumors, but numerous obstacles must be overcome to achieve the success observed in hematologic cancers. Potential solutions driven by advances in genetic engineering, synthetic biology, T cell biology, and improved tumor models that recapitulate the obstacles in human tumors are discussed., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Peptides that are presented by MHC class I (MHC-I) are processed from two potential sources, as follows: newly synthesized endogenous proteins for direct presentation on the surface of most nucleated cells and exogenous proteins for cross-presentation typically by professional APCs. In this study, we present data that implicate the nonclassical HLA-F and open conformers of MHC-I expressed on activated cells in a pathway for the presentation of exogenous proteins by MHC-I. This pathway is distinguished from the conventional endogenous pathway by its independence from TAP and tapasin and its sensitivity to inhibitors of lysosomal enzymes, and further distinguished by its dependence on MHC-I allotype-specific epitope recognition for Ag uptake. Thus, our data from in vitro experiments collectively support a previously unrecognized model of Ag cross-presentation mediated by HLA-F and MHC-I open conformers on activated lymphocytes and monocytes, which may significantly contribute to the regulation of immune system functions and the immune defense.

Infection by human CMV induces expression of the cellular MHC class I-related chain A (MICA) and chain B (MICB) surface proteins, which function as ligands for the activating NKG2D receptor. Engagement of NKG2D triggers NK cells and costimulates Ag-specific effector CD8 alphabeta T cells. The potency of MHC class I-related chain-NKG2D in stimulating these anti-viral immune responses may be countered by a CMV-encoded transmembrane glycoprotein, UL16, which specifically binds MICB as well as two of the UL16-binding proteins that are ligands of NKG2D. However, the function and significance of these interactions are undefined. Using a stably transfected B cell line, we show that expression of UL16 results in loss of surface MICB. This effect is caused by the failure of newly synthesized MICB to mature and transit the secretory pathway due to physical association with UL16. The intracellular retention of these protein complexes is mediated by a tyrosine-based motif in the cytoplasmic tail sequence of UL16, which determines localization to or retrieval from the trans-Golgi network. Deletion of this motif restores surface expression of MICB, whereas UL16 may be redirected to endosomal compartments. Predictably, the retention of MICB abrogates the stimulatory function of NKG2D. These results suggest a potential mechanism of viral immune evasion. However, this activity remains to be confirmed with CMV-infected fibroblasts or endothelial cells, in particular because MICB is normally coexpressed with MICA, which is not retained by UL16.

Due to their potent immunostimulatory capacity, dendritic cells (DC) have become the centerpiece of many vaccine regimens. Immature DC (DCimm) capture, process, and present Ags to CD4(+) lymphocytes, which reciprocally activate DCimm through CD40, and the resulting mature DC (DCmat) loose phagocytic capacity, but acquire the ability to efficiently stimulate CD8(+) lymphocytes. Recombinant vaccinia viruses (rVV) provide a rapid, easy, and efficient method to introduce Ags into DC, but we observed that rVV infection of DCimm results in blockade of DC maturation in response to all activation signals, including CD40L, monocyte-conditioned medium, LPS, TNF-alpha, and poly(I:C), and failure to induce a CD8(+) response. By contrast, DCmat can be infected with rVV and induce a CD8(+) response, but, having lost phagocytic activity, fail to process the Ag via the exogenous class II pathway. To overcome these limitations, we used the CMV protein pp65 as a model Ag and designed a gene containing the lysosomal-associated membrane protein 1 targeting sequence (Sig-pp65-LAMP1) to target pp65 to the class II compartment. DCmat infected with rVV-Sig-pp65-LAMP1 induced proliferation of pp65-specific CD4(+) clones and efficiently induced a pp65-specific CD4(+) response, suggesting that after DC maturation the intracellular processing machinery for class II remains intact for at least 16 h. Moreover, infection of DCmat with rVV-Sig-pp65-LAMP1 resulted in at least equivalent presentation to CD8(+) cells as infection with rVV-pp65. These results demonstrate that despite rVV interference with DCimm maturation, a single targeting vector can deliver Ags to DCmat for the effective simultaneous stimulation of both CD4(+) and CD8(+) cells.

The mammalian Y chromosome encodes male-specific minor histocompatibility (H-Y) Ags that are recognized by female T cells in an MHC-restricted manner. Two human H-Y epitopes presented by HLA-A2 and HLA-B7, respectively, have been identified previously and both are derived from the SMCY gene. We previously isolated CD8+ CTL clones that recognized a male-specific minor histocompatibility Ag presented by HLA-B8. In contrast to the SMCY-encoded H-Y epitopes, the B8/H-Y Ag was not presented by fibroblasts from male donors, suggesting that it was encoded by a novel gene. We now report that the HLA-B8-restricted H-Y epitope is defined by the octameric peptide LPHNHTDL corresponding to aa residues 566-573 of the human UTY protein. Transcription of the UTY gene is detected in a wide range of human tissues, but presentation of the UTY-derived H-Y epitope to CTL by cultured human cells shows significant cell-type specificity. Identification of this CTL-defined H-Y epitope should facilitate analysis of its contribution to graft/host interactions following sex-mismatched organ and bone marrow transplantation.

Adoptive immunotherapy of virus infection with viral-specific CTL has shown promise in animal models and human virus infections and is being evaluated as a therapy for established HIV-1 infection. Defining the individual obstacles for success is difficult in human trials. We have therefore examined the localization, persistence, and antiviral activity of HIV-1 gag-specific CTL clones in both HIV-1-infected and uninfected haplotype-matched human (hu)-PBL-SCID mice. Injection of gag-specific clones but not control CTL into HIV-1-infected hosts reduced plasma viremia by >10-fold but failed to eliminate the virus infection from most treated animals. The failure to eradicate virus did not reflect selection of escape variants because the gag epitope remained unmutated in virus isolates obtained after CTL therapy. Injection of carboxyfluorescein diacetate succinimide ester-labeled CTL demonstrated markedly different fates for gag-specific CTL in the presence or absence of HIV-1 infection. HIV-1-specific CTL rapidly disappeared in infected recipients, whereas they were maintained at high numbers in uninfected mice. By contrast, control CTL were long lived in both infected and uninfected recipients. Thus, interaction of CTL with virus-infected target cells in vivo leads not only to target destruction but also to the rapid disappearance of the infused CTL, and it limits the capacity of CTL therapy to eliminate HIV-1 infection.

The identification of Ags expressed by tumor cells and recognized by autologous T cells has led to the prospect of treating cancer by adoptive transfer of tumor-reactive T cells selected for Ag specificity. Tyrosinase is an Ag expressed by normal melanocytes as well as melanoma cells for which responses by autologous T cells have been detected. To evaluate the frequency with which tyrosinase-specific T cells can be isolated from melanoma patients for potential use in therapy, a recombinant vaccinia virus expressing tyrosinase was constructed for infection of autologous APCs that could be used to stimulate T cells reactive with this protein. Eight patients were studied, with peripheral blood serving as the source of both responder T cells and autologous APCs. Tyrosinase-specific CD8+ CTL clones were isolated from five of the eight patients with melanoma. The tyrosinase-specific CTL generated in this manner recognized autologous tumor cells as well as targets expressing the recombinant virus vector. CTL clones from three of the individuals were restricted to HLA-A28, -B8, and -B60, which have not previously been identified as alleles that can present immunogenic tyrosinase peptides. Tyrosinase-specific CD4+ T cell clones were isolated from six of the eight patients by stimulation with autologous APCs infected with recombinant vaccinia virus, and all these CD4+ clones were capable of recognizing autologous tumor cells. These studies demonstrate a high prevalence of CD4+ and CD8+ tyrosinase-specific responses in peripheral blood and support the feasibility of using peripheral blood to generate T cells for tumor therapy without the requirement for isolating T cells that have infiltrated tumor sites.

The characterization of the epitopes recognized by CTL provides insights into the nature of protective immune responses and facilitates the development of methods to enhance immunity to human pathogens. However, no easily applicable approach for CTL epitope identification has been developed. We present a rapid and efficient method for locating CTL epitopes within a protein. The gene encoding the protein of interest is inserted into an inducible prokaryotic expression vector. Random peptides are then generated by alkali digestion of intact or lysed Escherichia coli expressing the protein and assayed for the presence of the epitope by coating target cells for a standard CTL targeting assay. A large panel of clones containing serial 3'-deletions of the gene is then generated by exonuclease III digestion, and the expressed truncated proteins are similarly analyzed for the presence of the antigenic peptide. The epitope is located by determining the deletion points of clones expressing sequential truncations and differing in Ag expression. This technique was used to identify the H-2Ld-restricted nonamer in E. coli beta-galactosidase, with residues 876-884 representing the naturally processed epitope. To test the applicability of this method to other proteins, two genes from human CMV, an often fatal pathogen in immunocompromised individuals, were screened for HLA class I-restricted epitopes. An HLA-B18-restricted epitope from the CMV major immediate-early protein was found to lie between residues 378 and 389, and an HLA-B35-restricted epitope from the CMV pp65 matrix protein was characterized as residues 123 to 131. The results demonstrate that this technique can be used to rapidly identify CTL epitopes within a chosen protein and should be useful for assaying viral isolates or neoplasms for loss of epitopes after mutation and selection by host immune responses.

The therapeutic efficacy of adoptive immunotherapy of cancer has been shown to positively correlate with the dose of tumor-immune T cells transferred. Therefore, the success of this therapy is critically dependent on the ability to procure large numbers of functionally active T cells. Previous studies in animal models have shown that the limited therapeutic efficacy of a small number of immune T cells can be greatly enhanced by expansion of T cells in vitro to greater numbers before transfer in vivo. Optimal regimens for T cell expansion in vitro have generally employed the use of intermittent stimulation of the TCR with specific Ag followed by exogenous IL-2. The use of IL-2 alone does not provide for requisite episodic up-regulation of IL-2R. Stimulation of the invariant CD3 portion of the TCR/CD3 complex with antibody to CD3 (anti-CD3) represents an alternative method of up-regulating IL-2R and has been used to nonspecifically induce the growth of Ag-specific T cell lines and clones long-term in vitro with maintenance of function and specificity. The current study examined whether resting T cell populations containing small numbers of memory tumor-specific T cells could be rendered more effective in tumor therapy by nonspecific expansion in vitro with anti-CD3 plus IL-2. Spleens from C57BL/6 mice previously immunized to FBL-3, a syngeneic virus-induced leukemia, were nonspecifically stimulated with anti-CD3 plus IL-2. The resultant T cells were expanded in number, were nonlytic to FBL-3 but retained the ability to become lytic upon specific stimulation by FBL-3, and were effective in specific tumor therapy. The Ag-specific anti-tumor immune function declined on a per cell basis after each cycle of anti-CD3-induced T cell expansion. However, the approach resulted in a substantial increase in total T cell number and an overall net increase in the function of the effector T cell population. Thus, stimulation of tumor-immune T cell populations with anti-CD3 plus IL-2 represents a nonspecific method for expanding the number of specific effector T cells for cancer therapy.

The human pathogen CMV, is a major cause of morbidity and mortality in immunocompromised hosts. The CD8+ class I-restricted CTL response to CMV assists in preventing progression of CMV infection to life-threatening disease; however, the viral Ag recognized by CD8+ CTL are not well characterized. In general, virus-specific CTL recognize endogenously synthesized viral proteins processed and presented associated with class I MHC molecules. Although proteins or inactivated virions have been experimentally delivered to the cytoplasm to result in class I MHC presentation, this mode of Ag delivery to the class I processing pathway after natural viral entry has not been documented in humans. Our data demonstrate that the CMV-specific class I-restricted CTL response in individuals latently infected with CMV is predominantly specific for selected structural virion proteins introduced into the cell after viral penetration and efficient recognition occurs in the absence of de novo viral gene expression. This CTL response may provide a biological advantage for limiting the spread of infection after CMV reactivation because infected cells are lysed before viral assembly.