Intrinsic CD4+ T cell sensitivity and response to a pathogen are set and sustained by avidity for thymic and peripheral complexes of self peptide and MHC.

Interactions of T cell antigen receptors (TCRs) with complexes of self peptide and major histocompatibility complex (MHC) are crucial to T cell development, but their role in peripheral T cell responses remains unclear. Specific and nonspecific stimulation of LLO56 and LLO118 T cells, which transgenically express a TCR specific for the same Listeria monocytogenes epitope, elicited distinct interleukin 2 (IL-2) and phosphorylated kinase Erk responses, the strength of which was set in the thymus and maintained in the periphery in proportion to the avidity of the binding of the TCR to the self peptide-MHC complex. Deprivation of self peptide-MHC substantially compromised the population expansion of LLO56 T cells in response to L. monocytogenes in vivo. Despite their very different self-reactivity, LLO56 T cells and LLO118 T cells bound cognate peptide-MHC with an identical affinity, which challenges associations made between these parameters. Our findings highlight a crucial role for selecting ligands encountered during thymic 'education' in determining the intrinsic functionality of CD4+ T cells.

c-Myc-induced transcription factor AP4 is required for host protection mediated by CD8+ T cells.

Although the transcription factor c-Myc is essential for the establishment of a metabolically active and proliferative state in T cells after priming, its expression is transient. It remains unknown how T cell activation is maintained after c-Myc expression is downregulated. Here we identified AP4 as the transcription factor that was induced by c-Myc and sustained activation of antigen-specific CD8+ T cells. Despite normal priming, AP4-deficient CD8+ T cells failed to continue transcription of a broad range of c-Myc-dependent targets. Mice lacking AP4 specifically in CD8+ T cells showed enhanced susceptibility to infection with West Nile virus. Genome-wide analysis suggested that many activation-induced genes encoding molecules involved in metabolism were shared targets of c-Myc and AP4. Thus, AP4 maintains c-Myc-initiated cellular activation programs in CD8+ T cells to control microbial infection.

L-Myc expression by dendritic cells is required for optimal T-cell priming.

The transcription factors c-Myc and N-Myc--encoded by Myc and Mycn, respectively--regulate cellular growth and are required for embryonic development. A third paralogue, Mycl1, is dispensable for normal embryonic development but its biological function has remained unclear. To examine the in vivo function of Mycl1 in mice, we generated an inactivating Mycl1(gfp) allele that also reports Mycl1 expression. We find that Mycl1 is selectively expressed in dendritic cells (DCs) of the immune system and controlled by IRF8, and that during DC development, Mycl1 expression is initiated in the common DC progenitor concurrent with reduction in c-Myc expression. Mature DCs lack expression of c-Myc and N-Myc but maintain L-Myc expression even in the presence of inflammatory signals such as granulocyte-macrophage colony-stimulating factor. All DC subsets develop in Mycl1-deficient mice, but some subsets such as migratory CD103(+) conventional DCs in the lung and liver are greatly reduced at steady state. Importantly, loss of L-Myc by DCs causes a significant decrease in in vivo T-cell priming during infection by Listeria monocytogenes and vesicular stomatitis virus. The replacement of c-Myc by L-Myc in immature DCs may provide for Myc transcriptional activity in the setting of inflammation that is required for optimal T-cell priming.

Force-Regulated In Situ TCR-Peptide-Bound MHC Class II Kinetics Determine Functions of CD4+ T Cells.

We have recently shown that two-dimensional (2D) and force-regulated kinetics of TCR-peptide-bound MHC class I (pMHC-I) interactions predict responses of CD8(+) T cells. To test whether these findings are applicable to CD4(+) T cells, we analyzed the in situ 3.L2 TCR-pMHC-II interactions for a well-characterized panel of altered peptide ligands on the T cell surface using the adhesion frequency assay with a micropipette and the thermal fluctuation and force-clamp assays with a biomembrane force probe. We found that the 2D effective TCR-pMHC-II affinity and off-rate correlate with, but better predict the T cell response than, the corresponding measurements with the surface plasmon resonance in three dimensions. The 2D affinity of the CD4 for MHC-II was very low, approaching the detection limit, making it one to two orders of magnitude lower than the affinity of CD8 for MHC-I. In addition, the signal-dependent cooperation between TCR and coreceptor for pMHC binding previously observed for CD8 was not observed for CD4. Interestingly, force elicited TCR-pMHC-II catch-slip bonds for agonists but slip-only bonds for antagonists, thereby amplifying the power of discrimination between altered peptide ligands. These results show that the force-regulated 2D binding kinetics of the 3.L2 TCR for pMHC-II determine functions of CD4(+) T cells.

Distinct CD4+ helper T cells involved in primary and secondary responses to infection.

Helper T cells are critical for protective immunity, CD8(+) T-cell memory, and CD4(+) recall responses, but whether the same or distinct CD4(+) T cells are involved in these responses has not been established. Here we describe two CD4(+) T cells, LLO118 and LLO56, specific for an immunodominant Listeria monocytogenes epitope, with dramatically different responses to primary and secondary infection. Comparing in vivo responses, LLO118 T cells proliferate more strongly to primary infection, whereas surprisingly, LLO56 has a superior CD4(+) recall response to secondary infection. LLO118 T cells provide more robust help for CD8(+) T-cell responses to secondary infection than LLO56. We found no detectable differences in antigen sensitivity, but naive LLO118 T cells have much lower levels of CD5 and their T-cell receptor levels are dramatically down-regulated after their strong primary response. Thus, distinct CD4(+) helper T cells are specialized to help either in primary or secondary responses to infection.

Streptavidin-drug conjugates streamline optimization of antibody-based conditioning for hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) conditioning using antibody-drug conjugates (ADC) is a promising alternative to conventional chemotherapy- and irradiation-based conditioning regimens. The drug payload bound to an ADC is a key contributor to its efficacy and potential toxicities; however, a comparison of HSCT conditioning ADCs produced with different toxic payloads has not been performed. Indeed, ADC optimization studies in general are hampered by the inability to produce and screen multiple combinations of antibody and drug payload in a rapid, cost-effective manner. Herein, we used Click chemistry to covalently conjugate four different small molecule payloads to streptavidin; these streptavidin-drug conjugates can then be joined to any biotinylated antibody to produce stable, indirectly conjugated ADCs. Evaluating CD45-targeted ADCs produced with this system, we found the pyrrolobenzodiazepine (PBD) dimer SGD-1882 was the most effective payload for targeting mouse and human hematopoietic stem cells (HSCs) and acute myeloid leukemia cells. In murine syngeneic HSCT studies, a single dose of CD45-PBD enabled near-complete conversion to donor hematopoiesis. Finally, human CD45-PBD provided significant antitumor benefit in a patient-derived xenograft model of acute myeloid leukemia. As our streptavidin-drug conjugates were generated in-house with readily accessible equipment, reagents, and routine molecular biology techniques, we anticipate this flexible platform will facilitate the evaluation and optimization of ADCs for myriad targeting applications.

Antibody-drug conjugates plus Janus kinase inhibitors enable MHC-mismatched allogeneic hematopoietic stem cell transplantation.

Despite the curative potential of hematopoietic stem cell transplantation (HSCT), conditioning-associated toxicities preclude broader clinical application. Antibody-drug conjugates (ADCs) provide an attractive approach to HSCT conditioning that minimizes toxicity while retaining efficacy. Initial studies of ADC conditioning have largely focused on syngeneic HSCT. However, to treat acute leukemias or induce tolerance for solid organ transplantation, this approach must be expanded to allogeneic HSCT (allo-HSCT). Using murine allo-HSCT models, we show that pharmacologic Janus kinase 1/2 (JAK1/2) inhibition combined with CD45- or cKit-targeted ADCs enables robust multilineage alloengraftment. Strikingly, myeloid lineage donor chimerism exceeding 99% was achievable in fully MHC-mismatched HSCT using this approach. Mechanistic studies using the JAK1/2 inhibitor baricitinib revealed marked impairment of T and NK cell survival, proliferation, and effector function. NK cells were exquisitely sensitive to JAK1/2 inhibition due to interference with IL-15 signaling. Unlike irradiated mice, ADC-conditioned mice did not develop pathogenic graft-versus-host alloreactivity when challenged with mismatched T cells. Finally, the combination of ADCs and baricitinib balanced graft-versus-host disease and graft-versus-leukemia responses in delayed donor lymphocyte infusion models. Our allo-HSCT conditioning strategy exemplifies the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases.

CD4 Inhibits Helper T Cell Activation at Lower Affinity Threshold for Full-Length T Cell Receptors Than Single Chain Signaling Constructs.

CD4+ T cells are crucial for effective repression and elimination of cancer cells. Despite a paucity of CD4+ T cell receptor (TCR) clinical studies, CD4+ T cells are primed to become important therapeutics as they help circumvent tumor antigen escape and guide multifactorial immune responses. However, because CD8+ T cells directly kill tumor cells, most research has focused on the attributes of CD8+ TCRs. Less is known about how TCR affinity and CD4 expression affect CD4+ T cell activation in full length TCR (flTCR) and TCR single chain signaling (TCR-SCS) formats. Here, we generated an affinity panel of TCRs from CD4+ T cells and expressed them in flTCR and three TCR-SCS formats modeled after chimeric antigen receptors (CARs) to understand the contributions of TCR-pMHCII affinity, TCR format, and coreceptor CD4 interactions on CD4+ T cell activation. Strikingly, the coreceptor CD4 inhibited intermediate and high affinity TCR-construct activation by Lck-dependent and -independent mechanisms. These inhibition mechanisms had unique affinity thresholds dependent on the TCR format. Intracellular construct formats affected the tetramer staining for each TCR as well as IL-2 production. IL-2 production was promoted by increased TCR-pMHCII affinity and the flTCR format. Thus, CD4+ T cell therapy development should consider TCR affinity, CD4 expression, and construct format.

Comparison of Urine Antigen Assays for the Diagnosis of Histoplasma capsulatum Infection.

BACKGROUND: Urine antigen testing is a rapid sensitive method for detecting active infection with the endemic fungi Histoplasma capsulatum and Blastomyces dermatitidis. Herein, we compared the performance of the MiraVista Diagnostics (MVista) Histoplasma urine antigen assay with the Niche Diagnostics (ND) Histoplasma urine antigen assay for the detection of histoplasmosis and blastomycosis. METHODS: Two hundred fifty urine samples from 234 patients previously tested by the MVista Histoplasma urine antigen assay as part of routine care were tested by the ND Histoplasma and Blastomyces urine antigen assays. The electronic medical records of all patients whose samples were tested were retrospectively reviewed to identify patients with a clinical diagnosis of and/or treatment for histoplasmosis or blastomycosis, and the diagnostic workup undertaken to support these diagnoses. RESULTS: The MVista and ND Histoplasma urine antigen assays were highly concordant, showing 99% overall agreement (90.5% positive agreement and 99.6% negative agreement). Three specimens collected after antifungal therapy returned discrepant results, with the MVista assay positive in 2 of these and the ND assay positive in 1; in each case, the antigen concentration was near the lower quantification limit. Both Histoplasma assays were positive in all patients with culture-proven blastomycosis (n = 3). CONCLUSIONS: The MVista and ND Histoplasma urine antigen assays performed similarly in identifying histoplasmosis cases encountered in routine clinical practice, with discrepancies affecting posttreatment specimens. Given the paucity of Blastomyces-positive samples, further studies are needed to better compare the utility of the MVista and ND Histoplasma urine antigen assays in diagnosing blastomycosis.

Severe Cytomegalovirus Gastritis During Natalizumab-Mediated Immunosuppression.

We report a 35-year-old female receiving natalizumab as monotherapy for multiple sclerosis who subsequently developed severe cytomegalovirus gastritis. As cytomegalovirus gastritis has not been previously described during natalizumab treatment, we discuss the biological plausibility of this potential association and avenues for further study.

Accelerated humoral renal allograft rejection due to HLA-C14 mediated allosensitization to HLA-Bw6.

OBJECTIVES: To investigate immunological mechanisms underlying accelerated antibody-mediated rejection (AMR) of a living-related renal allograft in a patient with no detectable antibodies to donor human leukocyte antigens (HLA) in pre-transplant sera. METHODS: Pre- and post-transplant HLA antibody specificities were determined by single-antigen bead assay, and crossmatching was performed by flow cytometry- and complement-dependent cytotoxicity-based methods. Intermediate- and high-resolution HLA typing were performed by molecular methods. RESULTS: Pre-transplant patient serum reacted weakly against Bw6-positive beads; cytotoxicity and flow crossmatches were negative. The patient was mismatched for the donor antigens B62 and C10 (Bw6-positive). Following transplantation, strong antibody responses against B62, C10, and all Bw6-positive beads were detected. This reactivity was initially masked by complement interference, but became apparent at 1:20 dilution. High-resolution typing suggested that the anti-C16 antibody reactivity detected was an allele-specific response to donor C∗16:01 (Bw6-positive) but not recipient C∗16:02 (Bw6-negative). Alloimmunization likely occurred during pregnancy, during which HLA-C14 (Bw6-positive) was the only mismatched paternal HLA Class I allele. CONCLUSIONS: Sensitization to HLA-Bw6 via exposure to paternal HLA-C14 during pregnancy likely predisposed this patient to AMR. The case demonstrates the immunogenicity of HLA-C14-associated Bw6 epitopes in vivo and the clinical significance of low-level antibodies to HLA-Bw6.

High-affinity T cell receptor differentiates cognate peptide-MHC and altered peptide ligands with distinct kinetics and thermodynamics.

Interactions between the T cell receptor and cognate peptide-MHC are crucial initiating events in the adaptive immune response. These binding events are highly specific yet occur with micromolar affinity. Even weaker interactions between TCR and self-pMHC complexes play critical regulatory roles in T cell development, maintenance and coagonist activity. Due to their low-affinity, the kinetics and thermodynamics of such weak interactions are difficult to study. In this work, we used M15, a high-affinity TCR engineered from the 3.L2 TCR system, to study the binding properties, thermodynamics, and specificity of two altered peptide ligands (APLs). Our affinity measurements of the high-affinity TCR support the view that the wild type TCR binds these APLs in the millimolar affinity range, and hence very low affinities can still elicit biological functions. Finally, single methylene differences among the APLs gave rise to strikingly different binding thermodynamics. These minor changes in the pMHC antigen were associated with significant and unpredictable changes in both the entropy and enthalpy of the reaction. As the identical TCR was analyzed with several structurally similar ligands, the distinct thermodynamic binding profiles provide a mechanistic perspective on how exquisite antigen specificity is achieved by the T cell receptor.