The future of immunopsychiatry: Three milestones to clinical innovation.

Psychoneuroimmunology, the area of research dedicated to understanding the fundamental interactions between the central nervous system and the immune system, has given rise to the development of Immunopsychiatry, a new discipline which harnesses the immune system to produce beneficial outcomes for mental health problems. Immunopsychiatry has the potential to become a clinically relevant specialty area in psychiatric practice, but has not yet been adopted by the wider mental health community. This paper aims to map out the future trajectory of Immunopsychiatry on its road towards science-to-policy knowledge translation and clinical implementation. Three critical milestones which will need to be reached in order for Immunopsychiatry to fulfil its promise for clinical innovation are discussed: a clear definition of patients who fall within the immunopsychiatric continuum; demonstration of well-defined clinical benefit and incorporation in clinical guidelines; and convergence with other paradigms in biological psychiatry.

MR1-restricted mucosal-associated invariant T (MAIT) cells respond to mycobacterial vaccination and infection in nonhuman primates.

Studies on mucosal-associated invariant T cells (MAITs) in nonhuman primates (NHP), a physiologically relevant model of human immunity, are handicapped due to a lack of macaque MAIT-specific reagents. Here we show that while MR1 ligand-contact residues are conserved between human and multiple NHP species, three T-cell receptor contact-residue mutations in NHP MR1 diminish binding of human MR1 tetramers to macaque MAITs. Construction of naturally loaded macaque MR1 tetramers facilitated identification and characterization of macaque MR1-binding ligands and MAITs, both of which mirrored their human counterparts. Using the macaque MR1 tetramer we show that NHP MAITs activated in vivo in response to both Bacillus Calmette-Guerin vaccination and Mycobacterium tuberculosis infection. These results demonstrate that NHP and human MR1 and MAITs function analogously, and establish a preclinical animal model to test MAIT-targeted vaccines and therapeutics for human infectious and autoimmune disease.

Expanded tissue targets for foamy virus replication with simian immunodeficiency virus-induced immunosuppression.

Foamy viruses (FV) are the oldest known genus of retroviruses and have persisted in nonhuman primates for over 60 million years. FV are efficiently transmitted, leading to a lifelong nonpathogenic infection. Transmission is thought to occur through saliva, but the detailed mechanism is unknown. Interestingly, this persistent infection contrasts with the rapid cytopathicity caused by FV in vitro, suggesting a host defense against FV. To better understand the tissue specificity of FV replication and host immunologic defense against FV cytopathicity, we quantified FV in tissues of healthy rhesus macaques (RM) and those severely immunosuppressed by simian immunodeficiency virus (SIV). Contrary to earlier findings, we find that all immunocompetent animals consistently have high levels of viral RNA in oral tissues but not in other tissues examined, including the small intestine. Strikingly, abundant viral transcripts were detected in the small intestine of all of the SIV-infected RM, which has been shown to be a major site of SIV (and human immunodeficiency virus)-induced CD4+ T-cell depletion. In contrast, there was a trend to lower viral RNA levels in oropharyngeal tissues of SIV-infected animals. The expansion of FV replication to the small intestine but not to other CD4+ T-cell-depleted tissues suggests that factors other than T-cell depletion, such as dysregulation of the jejunal microenvironment after SIV infection, likely account for the expanded tissue tropism of FV replication.

Analysis of total human immunodeficiency virus (HIV)-specific CD4(+) and CD8(+) T-cell responses: relationship to viral load in untreated HIV infection.

Human immunodeficiency virus (HIV)-specific T-cell responses are thought to play a key role in viral load decline during primary infection and in determining the subsequent viral load set point. The requirements for this effect are unknown, partly because comprehensive analysis of total HIV-specific CD4(+) and CD8(+) T-cell responses to all HIV-encoded epitopes has not been accomplished. To assess these responses, we used cytokine flow cytometry and overlapping peptide pools encompassing all products of the HIV-1 genome to study total HIV-specific T-cell responses in 23 highly active antiretroviral therapy naïve HIV-infected patients. HIV-specific CD8(+) T-cell responses were detectable in all patients, ranging between 1.6 and 18.4% of total CD8(+) T cells. HIV-specific CD4(+) T-cell responses were present in 21 of 23 patients, although the responses were lower (0.2 to 2.94%). Contrary to previous reports, a positive correlation was identified between the plasma viral load and the total HIV-, Env-, and Nef-specific CD8(+) T-cell frequency. No correlation was found either between viral load and total or Gag-specific CD4(+) T-cell response or between the frequency of HIV-specific CD4(+) and CD8(+) T cells. These results suggest that overall frequencies of HIV-specific T cells are not the sole determinant of immune-mediated protection in HIV-infection.

Immunophenotypic analysis of hematogones (B-lymphocyte precursors) in 662 consecutive bone marrow specimens by 4-color flow cytometry.

Bone marrow hematogones (B-lymphocyte precursors) may cause problems in diagnosis because of their morphologic and immunophenotypic similarities to neoplastic lymphoblasts. The purposes of this prospective, multiparametric flow cytometry study were to quantify hematogones across age groups and a spectrum of clinical conditions, to identify factors that affect the relative quantity of hematogones, and to compare their immunophenotype with that of neoplastic lymphoblasts. A total of 662 consecutive marrow specimens were analyzed for hematogones using one of two 4-color antibody combinations; hematogones were identified in 528 (79.8%). There was a significant decline in hematogones with increasing age (P <.001), but a broad range was found at all ages and many adults had a relatively high number. Specimens processed by density gradient had a higher mean percent hematogones than those processed by erythrocyte lysis (P <.001). There was a direct decline in hematogones with increasing marrow involvement with neoplastic cells. A total of 8% of the 662 specimens contained 5% or more hematogones: 24.6% of specimens from patients aged less than 16 years and 6.3% from those 16 and older (P <.000 01). Increased hematogones were observed most often in patients with lymphoma, marrow regenerative states, immune cytopenias, and acquired immunodeficiency syndrome. Hematogones always exhibited a typical complex spectrum of antigen expression that defines the normal antigenic evolution of B-cell precursors and lacked aberrant expression. In contrast, lymphoblasts in 49 cases of precursor B-ALL showed maturation arrest and exhibited 1 to 11 immunophenotypic aberrancies. Four-color flow cytometry with optimal combinations of antibodies consistently distinguishes between hematogones and neoplastic lymphoblasts.

Immunophenotypic analysis of peripheral T-cell neoplasms. A multiparameter flow cytometric approach.

We retrospectively reviewed multiparameter flow cytometric analyses in 50 peripheral T-cell neoplasms (PTCNs). Results were interpreted within the context of a large cohort of nonneoplastic T-cell populations. All PTCN diagnoses were confirmed with morphologic and/or molecular analysis. Aberrant populations were defined as discrete immunophenotypic clusters exhibiting loss of or increased or diminished expression of T-cell antigens relative to internal immunophenotypically normal T-cell populations. An antigenic pattern was considered abnormal if it exceeded ranges for T-cell subsets in specific anatomic sites or was not normally encountered. Forty-six of 50 and 41 of 50 demonstrated 1 or more and 2 or more aberrations, respectively. The most common abnormally expressed antigen was CD3, followed by CD7, CD5, and CD2. Except for CD7, abnormally dim or bright antigen expression was more common than deletion. Only 3 cases were abnormal solely based on expansion of an otherwise immunophenotypically normal population; the remainder had patterns of antigen expression not seen in nonneoplastic populations. These data indicate that most PTCNs are aberrant by multiparameter flow analysis. However, results must be interpreted within the context of thorough knowledge of the immunophenotypic spectrum of nonneoplastic T cells.

CD4(+)CD8(dim) T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV-1 antigens.

CD4(+)CD8(dim) T cells represent a minor subset of the total CD3(+) T cell population in peripheral blood. Although transient and persistent expansions of these cells have been reported in both healthy and diseased individuals, the functional properties of the CD4(+)CD8(dim) population are largely unknown. In this study, we examined antigen-specific cytokine and proliferative responses of the CD4(+)CD8(dim) subset. In whole blood cultures stimulated with the viral antigens HCMV and HIV-1, a significant fraction of the CD4(+)CD8(dim) subset exhibited cytokine expression and proliferation in response to antigen activation. Typically, the CD4(+)CD8(dim) population contained two- to eightfold higher frequencies of antigen-specific cytokine producing cells than the CD4(+)CD8(-) population. Phenotypic analysis of the cytokine expressing CD4(+)CD8(dim) population indicated that these cells are memory T cells, with a high frequency of this population expressing the cytotoxic markers CD56 and perforin. Furthermore, the CD4(+)CD8(dim) cytokine responses to CMV were shown to be MHC class II dependent. Significantly, purified CD4(+)CD8(dim) T cells were found to possess higher CMV-specific cytotoxic activity than purified CD4(+)CD8(-) T cells in a standard (51)Cr-release CTL assay. Thus, CD4(+)CD8(dim) T cells appear to be MHC class II dependent, are capable of cytolytic effector activity, and are highly enriched within the CD4(+) cell populations specific for HCMV and HIV-1.

Use of overlapping peptide mixtures as antigens for cytokine flow cytometry.

Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.

Clonotypic structure of the human CD4+ memory T cell response to cytomegalovirus.

High steady-state frequencies of CMV-specific CD4(+) memory T cells are maintained in CMV-exposed subjects, and these cells are thought to play a key role in the immunologic control of this permanent infection. However, the essential components of this response are poorly defined. Here, we report the use of a step-wise application of flow cytometric and molecular techniques to determine the number and size of the TCR Vbeta-defined clonotypes within freshly obtained CMV-specific CD4(+) memory T cell populations of four healthy, CMV-exposed human subjects. This analysis revealed a stable clonotypic hierarchy in which 1-3 dominant clonotypes are maintained in concert with more numerous subdominant and minor clonotypes. These dominant clonotypes accounted for 10-50% of the overall CMV response, and comprised from 0.3 to 4.0% of peripheral blood CD4(+) T cells. Two subjects displayed immunodominant responses to single epitopes within the CMV matrix phosphoprotein pp65; these single epitope responses were mediated by a single dominant clonotype in one subject, and by multiple subdominant and minor clonotypes in the other. Thus, the CMV-specific CD4(+) T cell memory repertoire in normal subjects is characterized by striking clonotypic dominance and the potential for epitope focusing, suggesting that primary responsibility for immunosurveillance against CMV reactivation rests with a handful of clones recognizing a limited array of CMV determinants. These data have important implications for the understanding of mechanisms by which a genetically stable chronic viral pathogen such as CMV is controlled, and offer possible insight into the failure of such control for a genetically flexible pathogen like HIV-1.

Decay kinetics of human immunodeficiency virus-specific CD8+ T cells in peripheral blood after initiation of highly active antiretroviral therapy.

We measured the longitudinal responses to 95 HLA class I-restricted human immunodeficiency virus (HIV) epitopes and an immunodominant HLA A2-restricted cytomegalovirus (CMV) epitope in eight treatment-naive HIV-infected individuals, using intracellular cytokine staining. Patients were treated with highly active antiretroviral therapy (HAART) for a median of 78 weeks (range, 34 to 121 weeks). Seven of eight patients maintained an undetectable viral load for the duration of therapy. A rapid decline in HIV-specific CD8(+) T-cell response was observed at initiation of therapy. After an undetectable viral load was achieved, a slower decrease in HIV-specific CD8(+) T-cell response was observed that was well described by first-order kinetics. The median half-life for the rate of decay was 38.8 (20.3 to 68.0) weeks when data were expressed as percentage of peripheral CD8(+) T cells. In most cases, data were similar when expressed as the number of responding CD8(+) T cells per microliter of blood. In subjects who responded to more than one HIV epitope, rates of decline in response to the different epitopes were similar and varied by a factor of 2.2 or less. Discontinuation of treatment resulted in a rapid increase in HIV-specific CD8(+) T cells. Responses to CMV increased 1.6- and 2.8-fold within 16 weeks of initiation of HAART in two of three patients with a measurable CMV response. These data suggest that HAART quickly starts to restore CD8(+) T-cell responses to other chronic viral infections and leads to a slow decrease in HIV-specific CD8(+) T-cell response in HIV-infected patients. The slow decrease in the rate of CD8(+) T-cell response and rapid increase in response to recurrent viral replication suggest that the decrease in CD8(+) T-cell response observed represents a normal memory response to withdrawal of antigen.

Factors affecting the efficiency of CD8+ T cell cross-priming with exogenous antigens.

Processing of exogenous protein Ags by APC leads predominantly to presentation of peptides on class II MHC and, thus, stimulation of CD4+ T cell responses. However, "cross-priming" can also occur, whereby peptides derived from exogenous Ags become displayed on class I MHC molecules and stimulate CD8+ T cell responses. We compared the efficiency of cross-priming with exogenous proteins to use of peptide Ags in human whole blood using a flow cytometry assay to detect T cell intracellular cytokine production. CD8+ T cell responses to whole CMV proteins were poorly detected (compared with peptide responses) in most CMV-seropositive donors. Such responses could be increased by using higher doses of Ag than were required to achieve maximal CD4+ T cell responses. A minority of donors displayed significantly more efficient CD8+ T cell responses to whole protein, even at low Ag doses. These responses were MHC class I-restricted and dependent upon proteosomal processing, indicating that they were indeed due to cross-priming. The ability to efficiently cross-prime was not a function of the number of dendritic cells in the donor's blood. Neither supplementation of freshly isolated dendritic cells nor use of cultured, Ag-pulsed dendritic cells could significantly boost CD8 responses to whole-protein Ags in poorly cross-priming donors. Interestingly, freshly isolated monocytes performed almost as well as dendritic cells in inducing CD8 responses via cross-priming. In conclusion, the efficiency of cross-priming appears to be poor in most donors and is dependent upon properties of the individual's APC and/or T cell repertoire. It remains unknown whether cross-priming ability translates into any clinical advantage in ability to induce CD8+ T cell responses to foreign Ags.

Gamma interferon expression in CD8(+) T cells is a marker for circulating cytotoxic T lymphocytes that recognize an HLA A2-restricted epitope of human cytomegalovirus phosphoprotein pp65.

Antigen-specific CD8(+) T cells with cytotoxic activity are often critical in immune responses to infectious pathogens. To determine whether gamma interferon (IFN-gamma) expression is a surrogate marker for cytotoxic T lymphocytes (CTL), human cytomegalovirus-specific CTL responses were correlated with CD8(+) T-cell IFN-gamma expression determined by cytokine flow cytometry. A strong positive correlation was observed between specific lysis of peptide-pulsed targets in a (51)Cr release assay and frequencies of peptide-activated CD8(+) T cells expressing IFN-gamma at 6 h (r(2) = 0.72) or 7 days (r(2) = 0.91). Enumeration of responding cells expressing perforin, another marker associated with CTL, did not improve this correlation. These results demonstrate that IFN-gamma expression can be a functional surrogate for identification of CTL precursor cells.

The cutaneous response in humans to Treponema pallidum lipoprotein analogues involves cellular elements of both innate and adaptive immunity.

To extend prior studies implicating treponemal lipoproteins as major proinflammatory agonists of syphilitic infection, we examined the responses induced by intradermal injection of human subjects with synthetic lipoprotein analogues (lipopeptides) corresponding to the N termini of the 17- and 47-kDa lipoproteins of Treponema pallidum. Responses were assessed visually and by flow cytometric analysis of dermal leukocyte populations within fluids aspirated from suction blisters raised over the injection sites. Lipopeptides elicited dose-dependent increases in erythema/induration and cellular infiltrates. Compared with peripheral blood, blister fluids were highly enriched for monocytes/macrophages, cutaneous lymphocyte Ag-positive memory T cells, and dendritic cells. PB and blister fluids contained highly similar ratios of CD123(-)/CD11c(+) (DC1) and CD123(+)/CD11c(-) (DC2) dendritic cells. Staining for maturation/differentiation markers (CD83, CD1a) and costimulatory molecules (CD80/CD86) revealed that blister fluid DC1, but not DC2, cells were more developmentally advanced than their peripheral blood counterparts. Of particular relevance to the ability of syphilitic lesions to facilitate the transmission of M-tropic strains of HIV-1 was a marked enhancement of CCR5 positivity among mononuclear cells in the blister fluids. Treponemal lipopeptides have the capacity to induce an inflammatory milieu reminiscent of that found in early syphilis lesions. In contrast with in vitro studies, which have focused upon the ability of these agonists to stimulate isolated innate immune effector cells, in this study we show that in a complex tissue environment these molecules have the capacity to recruit cellular elements representing the adaptive as well as the innate arm of the cellular immune response.

De novo CD5+ diffuse large B-cell lymphomas. A heterogeneous group containing an unusual form of splenic lymphoma.

We reviewed our institutional experience with de novo CD5+, large B-cell lymphomas to determine whether they represent a distinct entity and are related to CD5+ small B-cell disorders. We identified 13 cases with multiparameter flow cytometry over a period of 58 months (5% of large B-cell lymphomas) in 7 females and 6 males. Three groups were identified. Group 1 (2 cases) had diffuse splenic red pulp involvement with a distinctive cordal pattern of infiltration, no other clinical evidence of mass disease, microscopic disseminated disease on further workup, and an identical immunoglobulin-negative immunophenotype. Group 2 cases (7 cases) were clinically and morphologically heterogeneous and had an immunophenotype resembling mantle cell lymphoma (FMC7-positive, CD23-). Group 3 (4 cases) had miscellaneous immunophenotypes, including one closely resembling chronic lymphocytic leukemia. Cyclin D1 was positive in only 1 of 10 evaluable cases (group 2). We conclude that CD5+ diffuse large B-cell lymphomas are heterogeneous; most cases do not seem to be related to chronic lymphocytic leukemia or mantle cell lymphoma. However, we identified a subgroup of primary splenic CD5+ large B-cell lymphoma with diffuse red pulp involvement and believe this may represent a distinct clinicopathologic entity.

Putative immunodominant human immunodeficiency virus-specific CD8(+) T-cell responses cannot be predicted by major histocompatibility complex class I haplotype.

Recent studies of human immunodeficiency virus (HIV)-specific CD8(+) T cells have focused on responses to single, usually HLA-A2-restricted epitopes as surrogate measures of the overall response to HIV. However, the assumption that a response to one epitope is representative of the total response is unconfirmed. Here we assess epitope immunodominance and HIV-specific CD8(+) T-cell response complexity using cytokine flow cytometry to examine CD8(+) T-cell responses in 11 HLA-A2(+) HIV(+) individuals. Initial studies demonstrated that only 4 of 11 patients recognized the putative immunodominant HLA-A2-restricted p17 epitope SLYNTVATL, suggesting that the remaining subjects might lack significant HIV-specific CD8(+) T-cell responses. However, five of six SLYNTVATL nonresponders recognized other HIV epitopes, and two of four SLYNTVATL responders had greater responses to HIV peptides restricted by other class I alleles. In several individuals, no HLA-A2-restricted epitopes were recognized, but CD8(+) T-cell responses were detected to epitopes restricted by other HLA class I alleles. These data indicate that an individual's overall CD8(+) T-cell response to HIV is not adequately represented by the response to a single epitope and that individual major histocompatibility complex class I alleles do not predict an immunodominant response restricted by that allele. Accurate quantification of total HIV-specific CD8(+) T-cell responses will require assessment of the response to all possible epitopes.

The CD4(+) T cell response to HIV-1.

Although virus-specific CD4(+) T cells have proven to be a critical component of the immunologic control of chronic viral infections in a number of models, the role and even the existence of HIV-1-specific CD4(+) T cells in human HIV-1 infection has been controversial. Recent advances in quantifying and functionally characterizing HIV-1-specific T cells may shed light on the participation of these cells in anti-HIV-1 host defense.

Identification of a human recent thymic emigrant phenotype.

The ability to measure human thymic output would be an invaluable tool for the study of the development of the naive T cell repertoire, as well as naive T cell regeneration after intensive cytotoxic chemotherapy or effective antiretroviral therapy of progressive HIV infection. We and others have demonstrated previously that quantification of T cell receptor rearrangement excision circles (TREC) within peripheral T cell populations provides insight into the frequency of recent thymic emigrants (RTE) and, therefore, into thymic function. However, measurement of RTE by this approach is complicated by the fact that TREC levels also are determined by turnover within the naive T cell compartment. Here, we report a phenotypic approach to RTE measurement. We demonstrate that alphaE integrin (CD103) expression is up-regulated very late in thymic development on a subset of CD8(+)/CD4(-) thymocytes and also defines a distinct subset of naive CD8(+) T cells in the periphery. The latter subset is differentiated from circulating CD103(+) mucosa-associated memory T cells by its naive T cell phenotype (CD45RO(-), CD62L(bright), CD27(bright), CD11a(dim), CD95(dim)) and its high concentration of TREC. Indeed, sorted CD103(+) naive CD8(+) cells display higher levels of TREC than their CD103(-) naive counterparts, and these cells demonstrate an age-related decline in frequency that is enhanced significantly by thymectomy. The thymic dependence of this subset and the cells' relatively evanescent presence in the periphery suggest that these cells are a population of RTE and that quantification of their frequency in peripheral blood provides an estimate of the level of ongoing thymopoiesis.

Immunofluorescence analysis of T-cell responses in health and disease.

The use of flow cytometry to study the functional responses of T cells by immunofluorescent staining for intracellular cytokines and other markers is a growing field of clinical interest. In this article, we describe methods for the rapid evaluation of T-cell responses to mitogens and specific antigens and explore how these assays might be valuable in various clinical settings.