T follicular helper (Tfh) CD4 cells are crucial providers of B cell help during adaptive immune responses. A circulating population of CD4 T cells, termed cTfh, have similarity to lymphoid Tfh, can provide B cell help, and responded to influenza vaccination. However, it is unclear whether human vaccination-induced cTfh respond in an antigen-specific manner and whether they form long-lasting memory. Here, we identified a cTfh population that expressed multiple T cell activation markers and could be readily identified by coexpression of ICOS and CD38. This subset expressed more Bcl-6, c-Maf, and IL-21 than other blood CD4 subsets. Influenza vaccination induced a strong response in the ICOS+CD38+ cTfh at day 7, and this population included hemagglutinin-specific cells by tetramer staining and antigen-stimulated Activation Induced Marker (AIM) expression. Moreover, TCRB sequencing identified a clonal response in ICOS+CD38+ cTfh that correlated strongly with the increased circulating ICOS+CD38+ cTfh frequency and the circulating plasmablast response. In subjects who received successive annual vaccinations, a recurrent oligoclonal response was identified in the ICOS+CD38+ cTfh subset at 7 days after every vaccination. These oligoclonal responses in ICOS+CD38+ cTfh after vaccination persisted in the ICOS−CD38− cTfh repertoire in subsequent years, suggesting clonal maintenance in a memory reservoir in the more-stable ICOS−CD38− cTfh subset. These data highlight the antigen-specificity, lineage relationships and memory properties of human cTfh responses to vaccination, providing new avenues for tracking and monitoring cTfh responses during infection and vaccination in humans.

Current paradigms of CD8 T cell-mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8 T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (T). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8 T cells in LTs also resemble T Moreover, high frequencies of HIV-specific CD8 T with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific T are enriched for effector-related immune genes and signatures compared with HIV-specific non-T in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8 T cell responses resident within LTs.

Follicular helper CD4+ T cells (TFH) play an integral role in promoting B cell differentiation and affinity maturation. While TFH cell frequencies are increased in lymph nodes (LN) from individuals infected with HIV, humoral immunity remains impaired during chronic HIV infection. Whether HIV inhibits TFH responses in LNs remains unclear. Advances in this area have been limited by the difficulty of accessing human lymphoid tissues. Here, we combined highdimensional mass cytometry with TCR repertoire sequencing to interrogate the composition of TFH cells in primary human LNs. We found evidence for intact antigen-driven clonal expansion of TFH cells and selective utilization of specific CDR3 motifs during chronic HIV infection, but the resulting TFH cells acquired an activation-related TFH cell signature characterized by IL-21 dominance. These IL-21+ TFH cells contained an oligoclonal HIV-reactive population, preferentially accumulated in patients with severe HIV infection, and associated with aberrant B cell distribution in the same LN. These data indicate that TFH cells remain capable of responding to HIV antigens during chronic HIV infection but become functionally skewed and oligoclonally restricted under persistent antigen stimulation.

The T-cell receptor (TCR) is required for maturation and function of regulatory T cells (Tregs), but the ligand specificities of Tregs outside the context of transgenic TCRs are largely unknown. Using peptide-MHC tetramers, we isolated rare specific Foxp3 + cells directly ex vivo from adult peripheral blood and defined their frequency and phenotype. We find that a proportion of circulating Tregs recognize foreign antigens and the frequency of these cells are similar to that of selfreactive Tregs in the absence of cognate infection. In contrast, the frequencies of Tregs that recognize some common microbial antigens are significantly reduced in the blood of most adults. Exposure to peripheral antigens likely has a major influence on the balance between Tregs and conventional T-cell subsets because a larger proportion of flu-specific T cells has a regulatory cell phenotype in the cord blood. Consistent with this finding, we show that lymphocytic choriomeningitis virus infection can directly modulate the ratio of virus-specific effectors and Tregs in mice. The resulting change in the balance within an antigen-specific T-cell population further correlates with the magnitude of effector response and the chronicity of infection. Taken together, our data highlight the importance of antigen specificity in the functional dynamics of the T-cell repertoire. Each specific population of CD4 + T cells in human peripheral blood contains a subset of Tregs at birth, but the balance between regulatory and effector subsets changes in response to peripheral antigen exposure and this could impact the robustness of antipathogen immunity.regulatory T cells | antigen specificity | repertoire | human | influenza

Graphical Abstract Highlights d High-dimensional profiling of CD4 + T cells in HIV-infected lymph nodes d A subset of CXCR5 À CD4 + T cells in lymph nodes are clonally related to T FH cells d CXCR5 À PD-1 + ICOS + CD4 + T cells exhibit T FH -like functional features d T FH -like CXCR5 À T cells contribute to circulating T cells with B cell help function In Brief Follicular helper T (T FH ) cells are critical for antibody production. Del Alcazar et al. showed that T FH cells can lose their characteristic chemokine receptor, giving rise to migratory populations of CXCR5 À T cells that retain B cell help function and are poised for CXCR5 expression.T FH cells B cells CXCR5 S1PR1 CCR2 S1P SUMMARYCXCR5 is a key marker of follicular helper T (T FH ) cells. Using primary lymph nodes (LNs) from HIVinfected patients, we identified a population of CXCR5 À CD4 + T cells with T FH -cell-like features. This CXCR5 À subset becomes expanded in severe HIV infection and is characterized by the upregulation of activation markers and high PD-1 and ICOS surface expression. Integrated analyses on the phenotypic heterogeneity, functional capacity, T cell receptor (TCR) repertoire, transcriptional profile, and epigenetic state of CXCR5 À PD-1 + ICOS + T cells revealed a shared clonal relationship with T FH cells. CXCR5 À PD-1 + ICOS + T cells retained a poised state for CXCR5 expression and exhibited a migratory transcriptional program. TCR sequence overlap revealed a contribution of LN-derived CXCR5 À PD-1 + ICOS + T cells to circulating CXCR5 À CD4 + T cells with B cell help function. These data link LN pathology to circulating T cells and expand the current understanding on the diversity of T cells that regulate B cell responses during chronic inflammation.

High dimensional profiling of activated CD4 + T cells in HIV infected lymph nodes revealed an accumulation of a CXCR5 negative subset.• CXCR5 -PD-1 + CD4 + T cells exhibited T FH -like protein expression and function.• CXCR5 -PD-1 + CD4 + T cells are related to T FH cells by clonal lineage and epigenetic similarity.• CXCR5 -PD-1 + CD4 + T cells upregulate a migratory gene program and contribute to circulating T cells with B cell help functionality Summary:CXCR5 is a key surface marker expressed on follicular helper T (T FH ) cells. We report here B cell help functionality in a population of CD4 + T cells isolated from primary human lymph nodes (LN) that lacked CXCR5expression. This CXCR5subset is distinguished from other CXCR5 -CD4 + T cells by high PD-1 expression.Accumulation of CXCR5 -PD-1 + T cells correlated with peripheral CD4 + T cell depletion and an increase in Tbet + B cells in the LN, highlighting these atypical CD4 + T cells as a key component of lymphoid dysregulation during chronic HIV infection. By interrogating the phenotypic heterogeneity, functional capacity, TCR repertoire, transcriptional profile, and epigenetic state of CXCR5 -PD-1 + T cells, we showed that CXCR5 -PD-1 + T cells are related to CXCR5 + PD-1 + T cells and provided evidence for the down regulation of CXCR5 following cell division as one mechanism for the absence of CXCR5 expression. Notably, CXCR5 -PD-1 + T cells exhibited a migratory transcriptional program and contributed to circulating CXCR5 -PD-1 + T cells with B cell help functionality in the peripheral blood. Thus, these data link LN pathology to circulating T cells and expand the current understanding on T cell diversity in the regulation of B cell responses during chronic inflammation.

Follicular-helper T cells (TFH) are an essential arm of the adaptive immune system. Although TFH were first discovered through their ability to contribute to antibody affinity maturation through co-stimulatory interactions with B cells, new light has been shed on their ability to remain a complex and functionally plastic cell type. Due to a lack sample availability, however, many studies have been limited to characterizing TFH in mice or non-canonical tissue types, such as peripheral blood. Such constraints have resulted in a limited, and sometimes contradictory, understanding of this fundamental cell type. One subset of TFH receiving attention in chronic infection are CXCR3-expressing TFH cells (CXCR3+TFH) due to their abnormal accumulation in secondary lymphoid tissues. Their function and clonal relationship with other TFH subsets in lymphoid tissues during infection, however, remains largely unclear. We thus systematically investigated this and other subsets of TFH within untreated HIV-infected human lymph nodes using Mass CyTOF and a combination of RNA and TCR repertoire sequencing. We show an inflation of the CXCR3+TFH compartment during HIV infection that correlates with a lower HIV burden. Deeper analysis into this population revealed a functional shift of CXCR3+TFH away from germinal center TFH (GC-TFH), including the altered expression of several important transcription factors and cytokines. CXCR3+TFH also upregulated cell migration transcriptional programs and were clonally related to peripheral TFH populations. In combination, these data suggest that CXCR3+TFH have a greater tendency to enter circulation than their CXCR3- counterparts, potentially functioning through distinct modalities that may lead to enhanced defense.