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Most cancer immunotherapies include activation of either innate or adaptive immune responses. We hypothesized that the combined activation of both innate and adaptive immunity will result in better antitumor efficacy. We have previously shown the synergy of an agonistic anti-CD40 mAb (anti-CD40) and CpG-oligodeoxynucleotides in activating macrophages to induce tumor cell killing in mice. Separately, we have shown that a direct intratumoral injection of immunocytokine (IC), an anti-GD2 Ab linked to IL-2, can activate T and NK cells resulting in antitumor effects. We hypothesized that activation of macrophages with anti-CD40/CpG, and NK cells with IC, would cause innate tumor destruction, leading to increased presentation of tumor Ags and adaptive T cell activation; the latter could be further augmented by anti-CTLA-4 Ab to achieve tumor eradication and immunological memory. Using the mouse GD2 + B78 melanoma model, we show that anti-CD40/CpG treatment led to upregulation of T cell activation markers in draining lymph nodes. Anti-CD40/CpG + IC/anti-CTLA-4 synergistically induced regression of advanced s.c. tumors, resulting in cure of some mice and development of immunological memory against B78 and wild type B16 tumors. Although the antitumor effect of anti-CD40/CpG did not require T cells, the antitumor effect of IC/anti-CTLA-4 was dependent on T cells. The combined treatment with anti-CD40/CpG + IC/anti-CTLA-4 reduced T regulatory cells in the tumors and was effective against distant solid tumors and lung metastases. We suggest that a combination of anti-CD40/CpG and IC/anti-CTLA-4 should be developed for clinical testing as a potentially effective novel immunotherapy strategy., Competing Interests: Dr. H. Loibner declares employment and ownership interests in Apeiron Biologics Inc. Dr. S. Gilles declares employment and ownership interests in Provenance Biopharmaceuticals. Dr. A. Korman declares employment and equity interests in Bristol Myers Squibb. All other authors declare no conflicts of interest., (Copyright © 2017 by The American Association of Immunologists, Inc.)

The programmed death-1 (PD-1) pathway limits the function of virus-specific T cells during chronic infection. We previously showed that blockade of the PD-1 pathway increases HIV-1-associated T cell function in vitro. However, the effect of PD-1 blockade on HIV-1 disease progression in vivo has not been examined. As in humans, HIV-1-infected humanized BALB/c-Rag2(-/-)γc(-/-) (Rag-hu) mice express elevated levels of PD-1 on T cells during chronic infection. To examine the effect of PD-1 blockade on disease progression, Rag-hu mice with chronic HIV-1 infection were treated with a blocking mAb directed against programmed cell death-1 ligand-1, the ligand for PD-1. Programmed cell death-1 ligand-1-treated Rag-hu mice exhibited a progressive decrease in the HIV-1 plasma viral load, with a 7-fold decrease by day 7, a 20-fold decrease by day 14, a 178-fold decrease by day 21, and a 269-fold decrease by day 28 postinitiation of treatment. By day 7, the percentage of CD4(+) T cells was statistically higher in the treated compared with the untreated group, and this trend was sustained throughout the 28-d treatment period. Moreover, there was a strong inverse correlation between plasma viral load and the percentage of both CD4(+) (r = -0.66; p < 0.0001) and CD8(+) (r = -0.64; p < 0.0001) T cells in the treated mice but not the untreated mice. This study provides "proof of concept" that humanized mice can be used to examine the effects of immunotherapeutic interventions on HIV-1 infection. Furthermore, to our knowledge, these data demonstrate for the first time that blockade of the PD-1 pathway reduces HIV-1 viral loads.

Allogeneic stem cell transplantation (allo-SCT) can cure hematological malignancies by inducing alloreactive T cell responses targeting minor histocompatibility antigens (MiHA) expressed on malignant cells. Despite induction of robust MiHA-specific T cell responses and long-term persistence of alloreactive memory T cells specific for the tumor, often these T cells fail to respond efficiently to tumor relapse. Previously, we demonstrated the involvement of the coinhibitory receptor programmed death-1 (PD-1) in suppressing MiHA-specific CD8(+) T cell immunity. In this study, we investigated whether B and T lymphocyte attenuator (BTLA) plays a similar role in functional impairment of MiHA-specific T cells after allo-SCT. In addition to PD-1, we observed higher BTLA expression on MiHA-specific CD8(+) T cells compared with that of the total population of CD8(+) effector-memory T cells. In addition, BTLA's ligand, herpes virus entry mediator (HVEM), was found constitutively expressed by myeloid leukemia, B cell lymphoma, and multiple myeloma cells. Interference with the BTLA-HVEM pathway, using a BTLA blocking Ab, augmented proliferation of BTLA(+)PD-1(+) MiHA-specific CD8(+) T cells by HVEM-expressing dendritic cells. Notably, we demonstrated that blocking of BTLA or PD-1 enhanced ex vivo proliferation of MiHA-specific CD8(+) T cells in respectively 7 and 9 of 11 allo-SCT patients. Notably, in 3 of 11 patients, the effect of BTLA blockade was more prominent than that of PD-1 blockade. Furthermore, these expanded MiHA-specific CD8(+) T cells competently produced effector cytokines and degranulated upon Ag reencounter. Together, these results demonstrate that BTLA-HVEM interactions impair MiHA-specific T cell functionality, providing a rationale for interfering with BTLA signaling in post-stem cell transplantation therapies.

Lymphocyte activation gene-3 (LAG-3; CD223) is a CD4 homolog that is required for maximal regulatory T cell function and for the control of CD4(+) and CD8(+) T cell homeostasis. Lag3(-)(/)(-) NOD mice developed substantially accelerated diabetes with 100% incidence. Adoptive transfer experiments revealed that LAG-3 was primarily responsible for limiting the pathogenic potential of CD4(+) T cells and, to a lesser extent, CD8(+) T cells. Lag3(-)(/)(-) mice exhibited accelerated, invasive insulitis, corresponding to increased CD4(+) and CD8(+) T cell islet infiltration and intraislet proliferation. The frequencies of islet Ag-reactive chromogranin A-specific CD4(+) T cells and islet specific glucose-6-phosphatase-specific CD8(+) T cells were significantly increased in the islets of Lag3(-)(/)(-) mice, suggesting an early expansion of pathogenic clones that is normally restrained by LAG-3. We conclude that LAG-3 is necessary for regulating CD4(+) and CD8(+) T cell function during autoimmune diabetes, and thus may contribute to limiting autoimmunity in disease-prone environments.

While T cells have been clearly implicated in a number of disease processes including autoimmunity, graft rejection, and atypical immune responses, the precise Ags recognized by the pathogenic T cells have often been difficult to identify. This has particularly been true for MHC class II-restricted CD4+ T cells. Although such cells can be demonstrated to have undergone clonal expansion at sites of pathology, they are frequently difficult to establish as stable T cell clones. Furthermore, in general, larger peptides in higher concentrations are required to stimulate CD4+ T cells than CD8+ T cells, which makes some of the techniques developed to identify CD8+ T cell Ags impractical. To circumvent some of these problems, we developed a model system consisting of two parts. The first part involves the construction of an indicator T cell hybridoma expressing a chimeric TCR comprised of murine constant regions and human variable regions specific for influenza hemagglutinin 307-319 presented by DR4. The second part consists of a library of fibroblasts each expressing multiple peptides as amino terminal covalent extensions of the beta-chain of HLA-DR4 (DRA1*0101, DRB1*0401). Using this model system, we screened approximately 100, 000 peptides and identified three novel peptides stimulatory for the HA1.7 TCR. While there is some convergence at residues known to be important for T cell recognition, all three peptides differ markedly from each other and bear little resemblance to wild-type hemagglutinin 307-319.