In Situ Characterization of Human Lymphoid Tissue Immune Cells by Multispectral Confocal Imaging and Quantitative Image Analysis; Implications for HIV Reservoir Characterization

CD4 T cells are key mediators of adaptive immune responses during infection and vaccination. Within secondary lymphoid organs, helper CD4 T cells, particularly those residing in germinal centers known as follicular helper T cells (Tfh), provide critical help to B-cells to promote their survival, isotype switching and selection of high affinity memory B-cells. On the other hand, the important role of Tfh cells for the maintenance of HIV reservoir is well documented. Thus, interrogating and better understanding the tissue specific micro-environment and immune subsets that contribute to optimal Tfh cell differentiation and function is important for designing successful prevention and cure strategies. Here, we describe the development and optimization of eight multispectral confocal microscopy immunofluorescence panels designed for in depth characterization and immune-profiling of relevant immune cells in formalin-fixed paraffin-embedded human lymphoid tissue samples. We provide a comprehensive library of antibodies to use for the characterization of CD4+ T-cells -including Tfh and regulatory T-cells- as well as CD8 T-cells, B-cells, macrophages and dendritic cells and discuss how the resulting multispectral confocal datasets can be quantitatively dissected using the HistoCytometry pipeline to collect information about relative frequencies and immune cell spatial distributions. Cells harboring actively transcribed virus are analyzed using an in-situ hybridization assay for the characterization of HIV mRNA positive cells in combination with additional protein markers (multispectral RNAscope). The application of this methodology to lymphoid tissues offers a means to interrogate multiple relevant immune cell targets simultaneously at increased resolution in a reproducible manner to guide CD4 T-cell studies in infection and vaccination

CD4 T Follicular Helper Cells and HIV Infection: Friends or Enemies?

Follicular T helper (Tfh) cells, a subset of CD4 T lymphocytes, are essential for memory B cell activation, survival, and differentiation and assist B cells in the production of antigen-specific antibodies. Work performed in recent years pointed out the importance of Tfh cells in the context of HIV and SIV infections. The importance of tissue distribution of Tfh is also an important point since their frequency differs between peripheral blood and lymph nodes compared to the spleen, the primary organ for B cell activation, and differentiation. Our recent observations indicated an early and profound loss of splenic Tfh cells. The role of transcriptional activator and repressor factors that control Tfh differentiation is also discussed in the context of HIV/SIV infection. Because Tfh cells are important for B cell differentiation and antibody production, accelerating the Tfh responses early during HIV/SIV infection could be promising as novel immunotherapeutic approach or alternative vaccine strategies. However, because Tfh cells are infected during the HIV/SIV infection and represent a reservoir, this may interfere with HIV vaccine strategy. Thus, Tfh represent the good and bad guys during HIV infection.JE from the Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS) and from The Canadian HIV Cure Enterprise Team Grant HIG-13305 from the Canadian Institutes of Health Research (CIHR) in partnership with CANFAR and IAS. FM is supported by a fellowship from Fondation du CHU de Québec. CB and YF are supported by fellowships from ANRS. JE acknowledges the support of the Canada Research Chair program. RS is supported by FCT—Fundaçao para a Ciência e a Tecnologia/MEC—Ministério da Educaçao e Ciência através de fundos nacionais e quando aplicavel cofinanciado pelo FEDER, no âmbito do Acordo de Parceria PT2020 referente à unidade de investigaçao n°4293. RS is supported by the Fundaçao para a Ciência e a Tecnologia (FCT) (IF/00021/2014)info:eu-repo/semantics/publishedVersio

Lymph Node Cellular Dynamics in Cancer and HIV: What Can We Learn for the Follicular CD4 (Tfh) Cells?

Lymph nodes (LNs) are central in the generation of adaptive immune responses. Follicular helper CD4 T (Tfh) cells, a highly differentiated CD4 population, provide critical help for the development of antigen-specific B cell responses within the germinal center. Throughout the past decade, numerous studies have revealed the important role of Tfh cells in Human Immunodeficiency Virus (HIV) pathogenesis as well as in the development of neutralizing antibodies post-infection and post-vaccination. It has also been established that tumors influence various immune cell subsets not only in their proximity, but also in draining lymph nodes. The role of local or tumor associated lymph node Tfh cells in disease progression is emerging. Comparative studies of Tfh cells in chronic infections and cancer could therefore provide novel information with regards to their differentiation plasticity and to the mechanisms regulating their development

PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection

Here, we report on the expression of programmed death (PD)-1 on human virus-specific CD8+ T cells and the effect of manipulating signaling through PD-1 on the survival, proliferation, and cytokine function of these cells. PD-1 expression was found to be low on naive CD8+ T cells and increased on memory CD8+ T cells according to antigen specificity. Memory CD8+ T cells specific for poorly controlled chronic persistent virus (HIV) more frequently expressed PD-1 than memory CD8+ T cells specific for well-controlled persistent virus (cytomegalovirus) or acute (vaccinia) viruses. PD-1 expression was independent of maturational markers on memory CD8+ T cells and was not directly associated with an inability to produce cytokines. Importantly, the level of PD-1 surface expression was the primary determinant of apoptosis sensitivity of virus-specific CD8+ T cells. Manipulation of PD-1 led to changes in the ability of the cells to survive and expand, which, over several days, affected the number of cells expressing cytokines. Therefore, PD-1 is a major regulator of apoptosis that can impact the frequency of antiviral T cells in chronic infections such as HIV, and could be manipulated to improve HIV-specific CD8+ T cell numbers, but possibly not all functions in vivo

Follicular CD4 T Helper Cells As a Major HIV Reservoir Compartment: A Molecular Perspective

Effective antiretroviral therapy (ART) has prevented the progression to AIDS and reduced HIV-related morbidities and mortality for the majority of infected individuals. However, a lifelong administration of ART is necessary, placing an inordinate burden on individuals and public health systems. Therefore, discovering therapeutic regimens able to eradicate or functionally cure HIV infection is of great importance. ART interruption leads to viral rebound highlighting the establishment and maintenance of a latent viral reservoir compartment even under long-term treatment. Follicular helper CD4 T cells (TFH) have been reported as a major cell compartment contributing to viral persistence, consequent to their susceptibility to infection and ability to release replication-competent new virions. Here, we discuss the molecular profiles and potential mechanisms that support the role of TFH cells as one of the major HIV reservoirs

Baseline Circulating Activated TFH and Tissue-Like Exhausted B Cells Negatively Correlate With Meningococcal C Conjugate Vaccine Induced Antibodies in HIV-Infected Individuals

Since 2006, meningococcal serogroup C (MenC) conjugate (MCC) vaccines have been supplied by the Brazilian government for HIV-infected children under 13 years old. For measuring protection against MenC, the serum bactericidal antibody (SBA) assay is the method of choice. The characterization of T follicular helper cells (TFH) cells has been an area of intensive study because of their significance in multiple human diseases and in vaccinology. The objective of this study was to characterize the phenotype of peripheral TFH cells and B cells and how they associated with each other and with SBA levels induced by vaccination as well as with serum cytokine levels of HIV-infected and non-infected children and adolescents. We found that CD27−IgD−CD21−CD38+ (exhausted B cells) as well as short-lived plasmablasts (CD27+IgD−CD21−CD38+) are increased in cART treated HIV patients and negatively associated with MCC vaccine induced SBA levels. Baseline frequency of activated peripheral TFH cells was a negative correlate for SBA response to MCC vaccine but positively correlated with circulating plasmablast frequency. Baseline IL4-levels positively associated with SBA response but showed a negative correlation with activated peripheral TFH cells frequency. The increased frequency of activated peripheral TFH cells found in non-responders to the vaccine implies that higher activation/differentiation of CD4 T cells within the lymph node is not necessarily associated with induction of vaccine responses

Preferential infection and depletion of Mycobacterium tuberculosis–specific CD4 T cells after HIV-1 infection

HIV-1 preferentially infects M. tuberculosis-specific CD4+ T cells due to their increased production of IL-2